Dye-labeled ribonucleotide triphosphates

ABSTRACT

The invention provides novel dye-labeled ribonucleotide analogs and methods for synthesizing those analogs. The compounds of the invention are especially useful for DNA sequencing by the polymerase chain reaction.

This application is a division of application Ser. No. 11/006,691, filedDec. 8, 2004, now U.S. Pat. No. 7,262,029, which is a division ofapplication Ser. No. 09/886,011, filed Jun. 22, 2001, now U.S. Pat. No.6,887,690, which are incorporated by reference herein.

The U.S. Government has a paid-up license in this invention and theright in limited circumstances to require the patent owner to licenseothers on reasonable terms as provided for by the terms of Award No.70NANB8H4002 awarded by the National Institute of Standards andTechnology (NIST) to the Perkin-Elmer Corp., Applied BiosystemsDivision.

INTRODUCTION

The development of reliable methods for determining the sequence of DNA(deoxyribonucleic acid) and RNA (ribonucleic acid) has been essential tothe success of recombinant DNA and genetic engineering. When used withthe other techniques of modern molecular biology, nucleic acidsequencing allows the dissection of animal, plant, and viral genomesinto discrete genes with defined chemical structures. Once a gene hasbeen isolated and characterized, it can be modified to produce desiredchanges in its sequence that allow the production of a gene product withproperties different from those of the original gene product.

The development of nucleic acid sequencing methods has involved paralleladvances in a variety of techniques. One was the emergence of simple andreliable methods for cloning small to medium-sized strands of DNA intobacterial plasmids, bacteriophages, and animal viruses. Cloning allowedthe production of pure DNA in sufficient quantities to allow chemicalanalysis. Another was the use of gel electrophoretic methods for thehigh resolution separation of oligonucleotides on the basis of size. Thekey development, however, was the introduction of methods of generatingsets of fragments of cloned, purified DNA that contain, in theircollection of lengths, the information necessary to define the sequenceof the nucleotides comprising the parent DNA molecules.

Presently, there are several approaches to determining the sequence of aDNA template, see, e.g., the dideoxy chain termination method, Sanger etal., Proc. Natl. Acad. Sci., 74:5463-67 (1977); the chemical degradationmethod, Maxam et al., Proc. Natl. Acad. Sci., 74:560-564 (1977); andhybridization methods, Drmanac et al., Genomics, 4:114-28 (1989),Khrapko, FEB 256:118-22 (1989). The most commonly used methods for DNAsequencing are based on the dideoxy chain termination method of Sangeret al., which involves enzymatic synthesis of single strands of DNA froma single stranded DNA template and a primer.

The basic dideoxy sequencing procedure involves (i) annealing anoligonucleotide primer to a template; (ii) extending the primer with DNApolymerase in four separate reactions, each containing a labelednucleotide or a labeled primer, a mixture of unlabeled dNTPs, and onechain-terminating ddNTP; (iii) resolving the four sets of reactionproducts by means of, for example, high-resolution denaturingpolyacrylamide/urea gel electrophoresis, capillary separation, or otherresolving means; and (iv) producing an autoradiographic image of the gelthat can be examined to infer the sequence. Alternatively, if adye-labeled primer or dideoxynucleotide is used, for example, inautomated sequencing procedures using Applied Biosystems Prism® 310,3100, 3700, or 377, the chain-terminated fragments can be resolved anddetected by fluorometry.

In the basic dideoxy chain termination method, four separate synthesesare carried out. In each, a single-stranded template is provided alongwith a primer, for example, a synthetic oligonucleotide or a restrictionfragment, that hybridizes to the template. In each of the foursequencing reactions, the primer is elongated at its 3′-terminus using aDNA polymerase in the presence of enough of a chain terminating analogof one of the four possible deoxynucleotides, for example, adideoxynucleotide (ddNTP), so that the growing chains are randomlyterminated by the 3′-incorporation of these “deadend” nucleotides. Theconcentration of chain terminating nucleotide relative to that ofdeoxynucleotides is adjusted to give a spread of termination eventscorresponding to all the possible chain lengths that can be resolved bygel electrophoresis. Enzymes currently used for this method ofsequencing include: the large fragment of Escherichia coli DNApolymerase I (“Klenow” fragment), reverse transcriptase, Thermusaquaticus (Taq) DNA polymerase, and a modified form of bacteriophage T7DNA polymerase (e.g., Sequenase®).

The four DNA synthesis reactions produce four sets of DNA products, eachproduct having one defined terminus and one variable terminus. Thedefined terminus starts with the primer molecule. The variable terminusends with a chain terminating agent specific for the nucleotide base(either G, A, T, or C) at which the synthesis reaction terminated. Thefour sets of products are each separated on the basis of their molecularweight, in four separate lanes of a high resolution polyacrylamide gel,to form four series of bands, with each band on the gel correspondingsequentially to a specific nucleotide in the DNA sequence. Thus, therelative positions of the bands identify the positions in the DNAsequence of each given nucleotide base. Generally, the DNA products arelabeled, for example, by including a radioactive nucleotide (e.g.,³⁵S-dATP, ³²P-dATP) in each reaction, so that the bands produced arereadily detected. Because the products from each of the four synthesisreactions must be run in separate gel lanes, problems frequently arisewhen comparing band mobilities between the different lanes.

The chain termination method has been modified in several ways, andserves as the basis for all currently available automated DNA sequencingmethods. See, e.g., Sanger et al., J. Mol. Biol., 143:161-78 (1980);Schreier et al., J. Mol. Biol., 129:169-72 (1979); Smith et al., NucleicAcids Research, 13:2399-2412 (1985); Smith et al., Nature, 321:674-79(1987), U.S. Pat. No. 5,171,534; Prober et al., Science, 238:33641(1987); Section II, Meth. Enzymol., 155:51-334 (1987); Church et al.,Science, 240:185-88 (1988); Swerdlow et al., Nucleic Acids Research, 18:1415-19 (1989); Ruiz-Martinez et al., Anal. Chem., 2851-58 (1993);Studier, PNAS, 86:6917-21 (1989); Kieleczawa et al., Science,258:1787-91; and Connell et al., Biotechniques, 5:342-348 (1987).

Two modifications of the original dideoxy method, which are commonlyused for automated DNA sequencing, are referred to as dye-primersequencing and dye-terminator sequencing. In dye-primer sequencing, afluorescently-labeled primer is used in combination with unlabeledddNTPs. The procedure requires four synthesis reactions and up to fourlanes on a gel for each template sequenced (i.e., one lane for each ofthe base-specific termination products). Following extension of thefluorescently-labeled primer, the sequencing reaction mixturescontaining ddNTP termination products are separated by electrophoresison a DNA sequencing gel. The size-separated, fluorescently-labeledproducts are automatically scanned with a laser at the bottom of the geland the fluorescence is detected with an appropriate monitor. (Smith etal., 1986, Nature 321:674-679, which is incorporated herein byreference). In a modification of this method, the primer added to eachof the four reactions is labeled with a different fluorescent marker.After the four separate sequencing reactions are completed, thereactions are combined and the mixture is subjected to gel analysis in asingle lane. The different fluorescent labels (one corresponding to eachof the four different base-specific termination products) areindividually detected.

Alternatively, in dye-terminator sequencing, a DNA polymerase is used toincorporate dNTPs onto the growing end of an unlabeled DNA primer untilthe enzyme incorporates a chain-terminating, fluorescently-labeled ddNTP(Lee et al., 1992, Nucleic Acid Research 20:2471). This process offersthe advantage of not having to synthesize dye-labeled primers. If eachdifferent ddNTP is labeled with a different fluorescent marker, all fourreactions can be performed in the same tube.

The availability of thermoresistant polymerases, such as Taq polymerase,has led to improved methods for sequencing, referred to as “cyclesequencing,” many of which are compatible with automated sequencingprotocols. See U.S. Pat. No. 5,075,216, which is incorporated herein byreference. In cycle sequencing, cycles of heating and cooling arerepeated allowing numerous extension products to be generated from eachmolecule of target. Murray, 1989, Nucleic Acids Research 17:8889, whichis incorporated herein by reference. The amplification of targetsequences complementary to the template sequence, in the presence ofdideoxy chain terminators, produces a family of extension products ofall possible lengths.

Because amplification of the template is part of the procedure, cyclesequencing, in theory, permits nucleotide sequence analysis to beperformed starting with small quantities of DNA. In practice, however,template quantity is often a limiting factor in cycle sequencing because(1) the amplification of chain-terminated templates is linear, notexponential as for full-length templates and (2) Taq DNA polymerasediscriminates against the incorporation of unconventional nucleotides,such as ddNTPs.

To achieve adequate incorporation of ddNTPs, DNA sequencing withthermostable DNA polymerases is performed using a mixture in which thechain-terminating nucleotide is present at a high concentration relativeto the concentration of the dNTPs, thus ensuring that a population ofextension products representing all possible fragment lengths over adistance of several hundred bases will be generated. Because ddNTPs areexpensive, cycle sequencing protocols achieve the desired ddNTP:dNTPconcentration ratio by using very low concentrations of the conventionaldNTPs. Such reaction mixtures create an environment wherein thethermostable polymerase is essentially starved for nucleotide substratesand DNA amplification is therefore very inefficient. Consequently, whenthe amount of template is below 10 ng per 100 bp, the cycle sequencingreaction is either very weak or fails completely.

Modified thermostable DNA polymerases having reduced discriminationagainst ddNTPs have been described. See European Patent No. 0 655 506A1; U.S. Pat. No. 5,614,365. One example of a modified thermostable DNApolymerase is the mutated form of T. aquaticus DNA polymerase having atyrosine residue at position 667 (instead of a phenylalanine residue),i.e. the F667Y mutated form of Taq DNA polymerase. AmpliTaq.RTM. FS,manufactured by Roche Diagnostics Corp. (Indianapolis, Ind.) andmarketed through Applied Biosystems, Inc. (Foster City, Calif.), reducesthe amount of ddNTP required for efficient nucleic acid sequencing of atarget by hundreds to thousands-fold. AmpliTaq.RTM. FS is a mutated formof T. aquaticus DNA polymerase having the F667Y mutation andadditionally an aspartic acid residue at position 46 (instead of aglycine residue; G46D mutation). Cycle sequencing methods using suchmutant DNA polymerases, however, still use chain terminators and, thus,do not offer the user the option of amplifying the reaction products ina geometric manner. When used to sequence small amounts of DNA startingmaterial, these methods, therefore, often require a first PCRamplification reaction and, then, a second cycle sequencing reaction.

Certain modified thermostable DNA polymerases have reduceddiscrimination against ribonucleotides. See U.S. Pat. No. 5,939,292,which is incorporated herein by reference. When these enzymes are usedin PCR methods, concentrations of dNTPs and/or rNTPs that are optimalfor target amplification may be employed. Unlike ddNTPs, incorporationof an rNTP does not result in a chain termination event, and, therefore,amplification in the presence of rNTPs is geometric.

SUMMARY OF THE INVENTION

The present invention provides dye-labeled ribonucleotides, which areuseful substrates for direct PCR sequencing (DPCRS) and other methodsinvolving the synthesis of labeled polynucleotides, which will beapparent to those of skill in the art. The dye-labeled ribonucleotideanalogs of the invention are efficiently incorporated into primerextension products by modified thermostable DNA polymerases. Thecompounds of the invention are useful in DNA sequencing, in detectingpolymorphisms, and in generating dye-labeled polynucleotides.

It is accordingly an object of the invention to provide methods forpreparing dye-labeled ribonucleotide analogs. It is a further object ofthe invention to provide methods for using dye-labeled ribonucleotideanalogs in improved, methods for DNA sequence analysis and for thepreparation of dye-labeled DNAs and RNAs, which may be used, forexample, as hybridization probes.

In one embodiment, the invention provides ribonucleotide analogs coupledto dyes through propargyl-ethyl-oxide-amino (EO) or propargylamino (PA)linkers.

In another embodiment, the invention provides dye-labeled purine analogshaving linkers that form intramolecular hydrogen bonds or chelates,thereby increasing the rigidity of the nucleotide analog and decreasingthe steric interactions between substituents linked at the C8 positionof a purine or the C7 position of a 7-deazapurine and5′-O-phosphorylated sugars. For purines, linkers at the C8 position formintramolecular hydrogen bonds or chelates with the correspondingnitrogen atom at position 7. For 7-deazapurines, linkers at C7 formintramolecular hydrogen bonds or chelates with the correspondingfunctional group at position 6.

In another embodiment, the invention provides dye-labeled pyrimidineanalogs having linkers at the C5 position that form intramolecularhydrogen bonds or chelates with the corresponding atom or atoms atposition 6, thereby altering the spatial orientation of the linker to,for example, enhance incorporation efficiency by a DNA polymerase.

In another embodiment, the invention provides dye-labeled purine analogshaving linkers at the C8 position that are covalently linked to the 5′carbon of the sugar, thereby locking the nucleoside in a conformationthat may favor incorporation by an polymerase enzyme.

In another embodiment, the invention provides dye-labeled pyrimidineanalogs having substituents at the C4 position that are covalentlylinked to the 5′ carbon of the sugar, thereby locking the nucleoside ina conformation that may favor incorporation by an polymerase enzyme.

In another embodiment, the invention provides dye-labeled ribonucleotideanalogs having tuned linkers that may reduce or eliminate mobilitydifferences between polynucleotides comprising different analogs, forexample, during polyacrylamide gel electrophoresis.

In another embodiment, the invention provides sets of dye-labeledribonucleotide analogs matched for peak height evenness and relativemobility during polyacrylamide gel electrophoresis.

In another embodiment, the invention provides a method for determiningthe sequence of a DNA template in a single reaction using sets ofdye-labeled ribonucleotide analogs.

In another embodiment, the invention provides a method for detectingsingle nucleotide polymorphisms (SNPs) using dye-labeled ribonucleotideanalogs.

In another embodiment, the invention provides a method for preparingrandomly-fragmented dye-labeled hybridization probes.

In another embodiment, the invention provides a method for preparingdye-labeled sense and antisense RNAs.

Additional objects and advantages of the invention will be set forth inpart in the description which follows, and in part will be apparent fromthe description, or may be learned by practice of the invention. Theobjects and advantages of the invention will be realized and attained bymeans of the elements and combinations particularly pointed out in theappended claims.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand do not limit or restrict the invention, as claimed.

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several embodiments of theinvention and together with the description, serve to explain theprinciples of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawing(s) will be provided by thePatent and Trademark Office upon request and payment of the necessaryfee.

FIG. 1 shows a method for detecting a single nucleotide polymorphism(SNP) using a mixture of dye-labeled rNTPs and dNTPs.

FIG. 2 shows a method for detecting a SNP on both strands of DNAtemplate simultaneously when one primer is longer than the other primer.

FIG. 3 shows a method for detecting a SNP on both strands of a DNAtemplate simultaneously using one primer with a modified base that willnot permit extension in the 5′ direction from point A.

FIG. 4 shows a method for detecting a SNP on both strands of a DNAtemplate simultaneously using hybridization based pull-out (HBP)primers.

DESCRIPTION OF THE EMBODIMENTS

The present invention provides novel compositions that are dye-labeledribonucleotide analogs, which may be incorporated in polynucleotides.Methods for using the ribonucleotide analogs of the invention for DNAsequencing, for detecting mutations, and for preparing dye-labeledpolynucleotides also are provided. The nucleotide analogs of theinvention enable the practice of novel methods for these purposes, whichare advantageous over prior procedures.

To facilitate the understanding of the invention, a number of terms aredefined below.

The term “nucleobase” means a nitrogen-containing heterocyclic moietycapable of forming Watson-Crick hydrogen bonds in pairing with acomplementary nucleobase or nucleobase analog, e.g., a purine, a7-deazapurine, or a pyrimidine. Typical nucleobases are the naturallyoccurring nucleobases adenine, guanine, cytosine, uracil, thymine, andanalogs of the naturally occurring nucleobases, e.g., 7-deazaadenine,7-deazaguanine, 7-deaza-8-azaguanine, 7-deaza-8-azaadenine, inosine,nebularine, nitropyrrole, nitroindole, 2-amino-purine,2,6-diamino-purine, hypoxanthine, pseudouridine, pseudocytidine,pseudoisocytidine, 5-propynyl-cytidine, isocytidine, isoguanine,7-deaza-guanine, 2-thio-pyrimidine, 6-thio-guanine, 4-thio-thymine,4-thio-uracil, O⁶-methyl-guanine, N⁶-methyl-adenine, O⁴-methyl-thymine,5, 6-dihydrothymine, 5,6-dihydrouracil, 4-methyl-indole, andethenoadenine (Fasman, Practical Handbook of Biochemistry and MolecularBiology, pp. 385-394, CRC Press, Boca Raton, Fla., 1989).

As used herein, “nucleoside” refers to a compound consisting of anucleobase linked to the C-1′ carbon of a ribose sugar. The ribose maybe substituted or unsubstituted. Substituted ribose sugars include, butare not limited to, those riboses in which one or more of the carbonatoms, for example the 2′-carbon atom, is substituted with one or moreof the same or different Cl, F, —R, —OR, —NR₂, or halogen groups, whereeach R is independently H, C₁-C₆ alkyl, or C₅-C₁₄ aryl. Ribose examplesinclude ribose, 2′-deoxyribose, 2′,3′-dideoxyribose, 2′-haloribose,2′-fluororibose, 2′-chlororibose, and 2′-alkylribose, e.g., 2′-O-methyl,4′-α-anomeric nucleotides, 1′-α-anomeric nucleotides, 2′-4′- and3′-4′-linked and other “locked” or “LNA”, bicyclic sugar modifications(WO 98/22489; WO 98/39352; WO 99/14226). LNA sugar analogs within anoligonucleotide are represented by the structures:

where B is any nucleobase.

Modifications at the 2′- or 3′-position of ribose include hydrogen,hydroxy, methoxy, ethoxy, allyloxy, isopropoxy, butoxy, isobutoxy,methoxyethyl, alkoxy, phenoxy, azido, amino, alkylamino, fluoro, chloro,and bromo. When the nucleobase is purine, e.g., A or G, the ribose sugaris attached to the N⁹-position of the nucleobase. When the nucleobase ispyrimidine, e.g., C, T or U, the pentose sugar is attached to theN¹-position of the nucleobase (Kornberg and Baker, DNA Replication,2^(nd) Ed., Freeman, San Francisco, Calif., 1992).

The term “nucleotide” refers to a phosphate ester of a nucleoside, as amonomer unit or within a nucleic acid. Nucleotides are sometimes denotedas “rNTP”, “NTP”, “dNTP” and “ddNTP” to particularly point out thestructural features of the ribose sugar, as illustrated by thestructures:

B is a nucleobase. “Ribonucleotide 5′-triphosphate” refers to rNTP, aribonucleotide with a triphosphate ester group at the 5′ position. Thetriphosphate ester group may include sulfur substitutions for thevarious oxygens, e.g., α-thio-nucleotide 5′-triphosphates. Nucleosidesand nucleotides include the natural D optical isomer, as well as the Loptical isomer forms (Garbesi (1993) Nucl. Acids Res. 21:4159-65;Fujimori (1990) J. Amer. Chem. Soc. 112:7435; Urata, (1993) NucleicAcids Symposium Ser. No. 29:69-70).

The terms “polynucleotide” or “oligonucleotide” are used hereininterchangeably and mean single-stranded and double-stranded polymers ofnucleotide monomers, including 2′-deoxyribonucleotides (DNA) andribonucleotides (RNA) linked by internucleotide phosphodiester bondlinkages, or internucleotide analogs, and associated counter ions, e.g.,H⁺, NH₄ ⁺, trialkylammonium, Mg²⁺, Na⁺, and the like. A polynucleotidemay be composed entirely of deoxyribonucleotides, entirely ofribonucleotides, or chimeric mixtures thereof. Polynucleotides may becomprised of nucleobase and sugar analogs. Polynucleotides typicallyrange in size from a few monomeric units, e.g., 5-40, when they arefrequently referred to in the art as oligonucleotides, to severalthousands of monomeric nucleotide units. Unless denoted otherwise,whenever a polynucleotide sequence is represented, it will be understoodthat the nucleotides are in 5′ to 3′ order from left to right and that“A” denotes deoxyadenosine, “C” denotes deoxycytidine, “G” denotesdeoxyguanosine, and “T” denotes thymidine.

The term “primer” as used herein refers to an oligonucleotide, whethernatural or synthetic, that is capable of acting as a point of initiationof nucleic acid synthesis under conditions in which primer extension isinitiated. The appropriate length of a primer depends on the intendeduse of the primer, but typically ranges from 15 to 35 nucleotides. Shortprimer molecules generally require cooler temperatures to formsufficiently stable hybrid complexes with the template. A primer neednot reflect the exact sequence of the template but must be sufficientlycomplementary to hybridize with a template for primer elongation tooccur.

Primers and nucleotides of the present invention may be labeled withmoieties that affect the rate of electrophoretic migration, i.e.mobility-modifying labels. Mobility-modifying labels include, but arenot limited to biotin, fluorescent dyes, cholesterol, andpolyethyleneoxy units, —(CH2CH2O)_(n)—, where n may be 1 to 100(Grossman et al., U.S. Pat. No. 5,624,800). Preferably, n is from 2 to20. The polyethyleneoxy (PEO) units may be interspersed with chargedgroups, such as phosphodiester to impart charge and increaseelectrophoretic mobility (velocity). The PEO label may be uncharged andact to retard electrophoretic or chromatographic mobility. Suchmodifiers may serve to influence or normalize the electrophoreticvelocity of amplification products during analysis, e.g., by fluorescentdetection, to improve resolution and separation (Grossman et al., U.S.Pat. No. 5,470,705).

Nucleotides labeled with a reporter dye and a mobility-modifier allowfor separation by electrophoresis of the primer extension fragments fromfragments without mobility-modifiers, substantially independent of thesize, i.e., number of nucleotides. That is, polynucleotides of the samelength may be discriminated by detection of spectrally resolvable dyelabels and separated on the basis of mobility-modifying labels.

Primers labeled with a mobility-modifier and used in primer extensionwith the reporter-labeled ribonucleotides of the invention generatefragments that may be separated from fragments generated from primersthat are not labeled. In this manner, opposing strands of adouble-stranded target nucleic acid may be sequenced.

Another class of labels serve to effect the separation or immobilizationof labeled fragments by specific or non-specific capture means, e.g.,biotin; 2,4-dinitrophenyl (DNP); and digoxigenin (Andrus, A. “Chemicalmethods for 5′ non-isotopic labelling of PCR probes and primers” (1995)in PCR 2: A Practical Approach, Oxford University Press, Oxford, pp.39-54).

As used herein, “alkyl” means a saturated or unsaturated, branched,straight-chain, branched, or cyclic hydrocarbon radical derived by theremoval of one hydrogen atom from a single carbon atom of a parentalkane, alkene, or alkyne. Typical alkyl groups consist of 1 to 12saturated and/or unsaturated carbons, including, but not limited to,methyl, ethyl, propyl, butyl, and the like.

As used herein, “alkyldiyl” means a saturated or unsaturated, branched,straight chain or cyclic hydrocarbon radical of 1 to 20 carbon atoms,and having two monovalent radical centers derived by the removal of twohydrogen atoms from the same or two different carbon atoms of a parentalkane, alkene or alkyne. Typical alkyldiyl radicals include, but arenot limited to, 1,2-ethyldiyl, 1,3-propyldiyl, 1,4-butyldiyl, and thelike.

As used herein, “aryl” means a monovalent aromatic hydrocarbon radicalof 6 to 20 carbon atoms derived by the removal of one hydrogen atom froma single carbon atom of a parent aromatic ring system. Typical arylgroups include, but are not limited to, radicals derived from benzene,substituted benzene, naphthalene, anthracene, biphenyl, and the like.

As used herein, the term “dye” refers to any reporter group whosepresence can be detected by its light absorbing or light emittingproperties. The term “dye” encompasses fluorescent compounds. Thefluorescent dyes of the invention include, but are not limited to,fluorescein-type dyes, rhodamine-type dyes, cyanine-type dyes, andEnergy transfer dye pairs.

The term “fluorescein-type dye” refers to a class of xanthene dyemolecules, which include the following substituted fused three-ringsystem:

where a wide variety of substitutions are possible at each ringposition. Fluorescein-type dyes are described, for example, in thefollowing U.S. Pat. No. 5,188,943., No. 5,654,442, No. 5,840,999, No.5,885,778, No. 6,008,379, No. 6,020,481, No. 6,096,723, and U.S. Pat.No. 6,221,604, each of which are hereby incorporated by referenceherein. Examples of fluorescein-type dyes useful as fluorescent labelsin DNA sequencing methods include, but are not limited to,6-carboxyfluorescein (6-FAM), 5-carboxyfluorescein (5-FAM), 5 or6-carboxy-4,7,2′,7′-tetrachlorofluorescein (TET), 5 or6-carboxy-4,7,2′,4′,5′,7′-hexachlorofluorescein (HEX), 5- or6-carboxy-4′,5′-dichloro-2′7′-dimethoxyfluorescein (JOE), and5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein (ZOE). Many times thedesignation -1 or -2 is placed after an abbreviation of a particulardye, e.g., HEX-1. The “-1” and “-2” designations indicate the particular5 or 6 dye isomer being used. The 1 and 2 isomers are defined by theelution order (the 1 isomer being the first to elute) of free dye in areverse-phase chromatographic separation system utilizing a C-8 columnand an elution gradient of 15% acetonitrile/85% 0.1 M triethylammoniumacetate to 35% acetonitrile/65% 0.1 M triethylammonium acetate.

The term “rhodamine-type dye” refers to a class of xanthene dyemolecules which include the following fused three-ring system:

where a wide variety of substitutions are possible at each ringposition. Rhodamine-type dyes are described, for example, in thefollowing U.S. Pat. No. 5,366,860, No. 5,840,999, No. 5,847,162, No.5,936,087, No. 6,008,379, No. 6,020,481, No. 6,025,505, No. 6,080,852,No. 6,111,116, No. 6,191,278, and U.S. Pat. No. 6,221,606, each of whichis incorporated by reference herein. Exemplary rhodamine-type dyesuseful as dye labels include, but are not limited to,tetramethylrhodamine (TAMRA), 4,7-dichlorotetramethyl rhodamine(dTAMRA), rhodamine X (ROX), 4,7-dichlororhodamine X (dROX), rhodamine6G (R6G), 4,7-dichlororhodamine 6G (dR6G), rhodamine 110 (R110),4,7-dichlororhodamine 110 (dR110), and the like (Bergot, et al., U.S.Pat. No. 5,366,860 (1994); Lee et al., Nucleic Acids Research, 20(10):2471-2483 (1992)), with the following structures:

For each of the above rhodamine-type dyes, X is the site of attachmentto the linker in the compounds of the invention.

The term “cyanine-type dye” refers to a class of dye molecules with thefollowing basic structure:

wherein A and B are independently C, O, S, or N, wherein a wide varietyof substitutions are possible at each position and wherein n isgenerally a number from 1 to 3, but may be larger. Exemplarycyanine-type dyes useful as dye labels include, but are not limited to,the compounds disclosed in the following U.S. Pat. No. 5,986,086, No.6,027,709, No. 6114,350, No. 6,150,107, No. 6,197,956, No. 6,224,644,and U.S. Pat. No. 6,225,050, each of which is incorporated by referenceherein.

The term “energy transfer dye pair” refers to a class of dyes in whichtwo fluorescent dyes are covalently attached. In general, a fluorescein,or other donor dye, is attached to a rhodamine, or other acceptor dye.Energy transfer dye pairs are described, for example, in the followingU.S. Pat. No. 5,800,996, Nos. 5,863,727, and 5,945,526, each of which ishereby incorporated by reference herein. An exemplary energy transferdye pair has the structure:

As used herein, the term “single nucleotide polymorphism” or “SNP”refers to DNA sequence variations that occur when a single nucleotide inthe genome sequence is altered. SNPs occur every 100 to 300 bases alongthe 3-billion-base human genome in both coding (gene) and noncodingregions of the genome.

As used herein, the term “DNA sequencing reaction mixture” refers to areaction mixture that comprises elements necessary for a DNA sequencingreaction. Thus, a DNA sequencing reaction mixture is suitable for use ina DNA sequencing method for determining the nucleic acid sequence of atarget, although the reaction mixture may initially be incomplete, sothat the initiation of the sequencing reaction is controlled by theuser. In this manner, the reaction may be initiated once a finalelement, such as the enzyme, is added, to provide a complete DNAsequencing reaction mixture. Typically, a DNA sequencing reaction willcontain a buffer suitable for polymerization activity, nucleosidetriphosphates, and at least one unconventional nucleotide, for example addNTP or an rNTP. The reaction mixture also may contain a primersuitable for extension on a target by a polymerase enzyme, a polymerase,and a target nucleic acid. Either the primer or one of the nucleotidesis generally labeled with a detectable moiety such as a fluorescentlabel. Generally, the reaction is a mixture that comprises fourconventional nucleotides and at least one unconventional nucleotide. Inone embodiment of the invention, the polymerase is a modifiedthermostable DNA polymerase and the unconventional nucleotide is adye-labeled ribonucleotide.

The term “polymerase” refers to an enzyme that catalyzes the initiationof synthesis and the elongation of a polynucleotide chain complementaryto a polynucleotide template. Polymerases may incorporateribonucleotides, deoxyribonucleotides, modified ribonucleotides,modified deoxyribonucleotides, or combinations into the elongatingchain. Polymerases may initiate polynucleotide synthesis either byrecognizing specific initiation sites in the template or by extending aprimer complementary to the template. Polymerases may have additionalenzymatic activities including, but not limited to, 5′-3′ exonucleaseactivity and 3′-5′ exonuxlease activity.

The term “thermostable polymerase” refers to an enzyme that is stable toheat, is heat resistant, and retains sufficient activity to effectsubsequent primer extension reactions when subjected to the elevatedtemperatures for the time necessary to effect denaturation ofdouble-stranded nucleic acids. Heating conditions necessary for nucleicacid denaturation are well known in the art and are exemplified in U.S.Pat. Nos. 4,683,202 and 4,683,195, which are incorporated herein byreference. As used herein, a thermostable polymerase is suitable for usein a temperature cycling reaction such as the polymerase chain reaction(“PCR”). For a thermostable polymerase, enzymatic activity refers to thecatalysis of the combination of the nucleotides in the proper manner toform primer extension products that are complementary to a templatenucleic acid strand.

The term “modified thermostable polymerase” refers to a thermostablepolymerase that has been altered, for example, chemically or usingrecombinant DNA techniques, so that it exhibits increased efficiency inthe incorporation of unconventional nucleotides.

The term “unconventional” when referring to a nucleobase, a nucleoside,or a nucleotide, includes any modification, derivations, or analogues ofconventional bases, or nucleotides that naturally occur in DNA, i.e.,dATP, dGTP, dCTP, and dTTP. For example, although for RNA the naturallyoccurring nucleotides are ribonucleotides (i.e., rATP, rGTP, rCTP, rUTPcollectively rNTPs), because these nucleotides have a hydroxyl group atthe 2′ position of the sugar, which, by comparison is absent in dNTPs,as used herein, ribonucleotides or analogues of ribonucleotides areunconventional nucleotides for incorporation in DNA. Similarly,dideoxynucleotides also are unconventional bases. Unconventionalnucleotides may be labeled with dyes, for example, fluorescein,rhodamine; dichlororhodamine (d-rhodamine), or cyanine dyes dyes.

As used herein, the term “linker” refers to moieties that form a linkagebetween the base portion of a ribonucleotide and a dye. Linkers usefulfor preparing the dye-labeled ribonucleotides of the invention include,but are not limited to moieties of the general formula:

wherein each of n, o, and p are integers ranging from 0 to 3 and the sumof n+o+p is at least 2, and wherein W, X, Y, and Z are independentlycarbon or nitrogen, as disclosed in U.S. Pat. Nos. 6,096,875 and6,197,555, which are hereby incorporated herein by reference. Linkerscomprising this structure are referred to herein as “rigid linkers.”Additional moieties useful as linkers in the invention include, but arenot limited to the following structures:

referred to herein as a “propargyl-ethyl-oxide-amino linker” or “EOlinker”;

referred to herein as a “propargyl-propyl-oxide-amino linker” or “POlinker”;

referred to herein as a “propargyl linker” or “PA linker”;

referred to herein as a “phosphate linker” or “P linker”;

referred to herein as a “benzylamine linker” or “Bn Linker.” The ring inbenzylamine linkers may be substituted in a chemically reasonablemanner.

Linker moieties useful in the invention include polymers of the abovestructures, for example, EO-Bn linkers, EO-P linkers, EO-P-Bn linkers,etc.

As used herein, the term “tuned linkers” refers to linkers that havealtered hydrophobicity or hydrophilicity, for example, because of theaddition of a side chain. Such linkers may be used, for example, toadjust the mobility of the dye-labeled ribonucleotides of the inventionduring polyacrylamide gel electrophoresis, to adjust the spectralproperties of dye-labeled ribonucleotides, to minimize quenching, or toalter the emission maxima of dye-labeled ribonucleotides. By tuning thedye-labeled ribonucleotide analogs corresponding to rATP, rGTP, rCTP,and rUTP, 4-color sets of the compounds of the invention having improvedrelative electrophoretic mobility and peak height evenness are providedfor use in DNA sequencing protocols.

As used herein, the term “heterocycle linker” refers to any linker thatcan form an intermolecular chelate, preferably 5 or 6-membered, withwater or any metal and the respective nucleobase.

As used herein, the term “independently” means that the identity of eachelement is selected without regard to the identity of other elements andmay be either the same or different from that of other elements.

As used herein, the term“multimer” refers to polymers of unit monomers,wherein the polymer may comprise monomers of a single type or ofdifferent types in any order or combination. Preferably, a multimerconsists of 2 to 10 monomers.

In order to further aid the understanding of the invention, specificdye-labeled ribonucleotide analogs are referred to throughout thespecification. These references are not, however, intended to limit thescope of the invention.

The dye-labeled ribonucleotides of the invention are compounds of thegeneral formula:

wherein Dye is any reporter group, preferably selected fromfluorescein-type dyes, rhodamine-type dyes, energy transfer dye pairs,and cyanine-type dyes;

wherein L is a linker, preferably a propargyl-ethyl-oxide-amino linker,a propargylamino linker, a propargyl-propyl-oxide-amino linker, abenzylamine linker, a phosphate linker, a rigid linker, a heterocyclelinker, a tuned linker, or a multimer of these linkers;

wherein B is a nucleobase;

wherein R₁ and R₂ are independently H, OH, NH₂, or SH, preferably OH;and

wherein R₃ is triphosphate, α-thiotri, or a salt thereof.

The dye-labeled ribonucleotides of the invention include pyrimidine-typecompounds of the general formula I:

-   -   wherein X is N, NH, or C;    -   wherein Y is O or NH₂;    -   wherein R₁ and R₂ are independently H, OH, NH₂, or SH,        preferably OH;    -   wherein R₃ is either triphosphate, α-thiotriphosphate, or a salt        thereof;    -   wherein L₁ is a linker, preferably a propargyl-ethyl-oxide-amino        linker, a propargylamino linker, a propargyl-propyl-oxide-amino        linker, a benzylamine linker, a phosphate linker, a rigid        linker, or a multimer of these linkers, more preferably either a        propargyl-ethyl-oxide-amino linker or a propargylamino linker;    -   wherein L₂ is a benzylamine linker or a phosphate linker;    -   wherein n=0-4, m=0-4, and m+n is at least 1; and;    -   wherein the dye is any reporter group, preferably a        rhodamine-type dye, a fluorescein-type dye, an energy transfer        dye, or a cyanine-type dye.

The dye-labeled ribonucleotides of the invention also includepurine-type compounds of the general formula II:

-   -   wherein L is a linker, preferably a propargyl-ethyl-oxide-amino        linker, propargylamino linker, propargyl-propyl-oxide-amino        linker, benzylamine linker, phosphate linker, or rigid linker,        more preferably either a propargyl-ethyl-oxide-amino linker or a        propargylamino linker;    -   wherein R₄ is either NH₂, OH, or O, and R₅ is either NH₂, OH, or        H;    -   wherein R₁ and R₂ are independently H, OH, NH₂, or SH,        preferably OH;    -   wherein R₃ is either triphosphate, α-thiotriphosphate, or a salt        thereof; and    -   wherein the dye is any reporter group, preferably a        rhodamine-type dye, a fluorescein-type dye, an energy transfer        dye, or a cyanine-type dye.

The dye-labeled ribonucleotides of the invention also includepurine-type compounds of the general formula III:

-   -   wherein L₁ is a linker, preferably a propargyl-ethyl-oxide-amino        linker, propargylamino linker, propargyl-propyl-oxide-amino        linker, benzylamine linker, phosphate linker, or rigid linker,        more preferably either a propargyl-ethyl-oxide-amino linker or a        propargylamino linker;    -   wherein L₂ is a benzylamine linker or a phosphate linker;    -   wherein n=0-4, m=0-4, and m+n is at least 1;    -   wherein R₄ is either NH₂, OH, or O, and R₅ is either NH₂, OH, or        H;    -   wherein R₁ and R₂ are independently H, OH, NH₂, or SH,        preferably OH;    -   wherein R₃ is either triphosphate, α-thiotriphosphate, or a salt        thereof; and    -   wherein the dye is any reporter group, preferably a        rhodamine-type dye, a fluorescein-type dye, a energy transfer        dye, or a cyanine-type dye.

In another embodiment, the dye-labeled ribonucleotides of the inventioninclude purine-type compounds of the general formula IV:

-   -   wherein R₁, R₂, and R₄ are independently H, O, OR, S, SR, NR₂,        or CR₂;    -   wherein R₃ is SR, NR₂, OR, or CR₂ and comprises a reporter        group, preferably a rhodamine-type dye, a fluorescein-type dye,        an energy transfer dye, or a cyanine-type dye;    -   wherein R is hydrogen, alkyl, preferably C1-C6 alkyl, C2-C6        alkenyl, or C2-C6 alkynyl, aryl, preferably benzyl substituted        at from 0 to 3 positions in a chemically reasonable manner with        F, Cl, Br, I, C1-C18 alkyl, Silyl, OH, OR′, SH, SR′, SOR′,        SO₂R′, SO₃, NH₂, NHR′, or NR′₂, or an amino acid;    -   wherein R₅ and R₆ are independently H, OH, NH₂, or SH,        preferably OH;    -   wherein R₇ is either triphosphate, α-thiotriphosphate, or a salt        thereof;    -   wherein R′ is OH, alkyl, preferably C1-C6 alkyl, or alkynyl,        preferably C2-C6 alkynyl;    -   wherein X, Y, and Z are independently carbon, nitrogen, oxygen,        sulfur, phosphorus, or selenium;    -   wherein n is 0 or 1; and    -   wherein M is H₂O or any metal, preferably a Group IA metal or a        Group IIA metal, more preferably Li⁺, Na⁺, K⁺, Mg²⁺, or Ca²⁺.

In formula IV, the linker moiety at C8 may be in the same plane as thepurine or skewed to lie outside of the purine plane.

In another embodiment, the dye-labeled ribonucleotides of the inventioninclude purine-type compounds of the general formula V:

-   -   wherein R₁, R₂, and R₄ are independently H, O, OR, S, SR, NR₂,        or CR₂,    -   wherein R₃ is SR, NR₂, OR, or CR₂ and comprises a reporter        group, preferably a rhodamine-type dye, a fluorescein-type dye,        an energy transfer dye, or a cyanine dye;    -   wherein R is hydrogen, alkyl, preferably C1-C6 alkyl, C2-C6        alkenyl, or C2-C6 alkynyl, aryl, preferably benzyl substituted        at from 0 to 3 positions in a chemically reasonable manner with        F, Cl, Br, I, C1-C18 alkyl, Silyl, OH, OR′, SH, SR′, SOR′,        SO₂R′, SO₃, NH₂, NHR′, or NR′₂, or an amino acid;    -   wherein R₅ and R₆ are independently H, OH, NH₂, or SH;    -   wherein R₇ is either triphosphate, α-thiotriphosphate, or a salt        thereof;    -   wherein R′ is OH, alkyl, preferably C1-C6 alkyl, or alkynyl,        preferably C2-C6 alkynyl;    -   wherein X, Y, and Z are independently C, N, O, S, P, or Se;    -   wherein n is 0 or 1; and    -   wherein M is H₂O or any metal, preferably a Group IA metal or a        Group IIA metal, more preferably Li⁺, Na⁺, K⁺, Mg²⁺, or Ca²⁺.

In formula V, the linker moiety at C7 may be in the same plane as thepurine or skewed to lie outside of the purine plane.

In another embodiment, the dye-labeled ribonucleotides of the inventioninclude pyrimidine-type compounds of the general formula VI:

-   -   wherein R₁ is H, O, OR, S, SR, NR₂, or CR₂,    -   wherein R₂ is SR, NR₂, OR, or CR₂ and comprises a reporter        group, preferably a rhodamine-type dye, a fluorescein-type dye,        an energy transfer dye, or a cyanine dye;    -   wherein R is hydrogen, alkyl, preferably C1-C6 alkyl, C2-C6        alkenyl, or C2-C6 alkynyl, aryl, preferably benzyl substituted        at from 0 to 3 positions in a chemically reasonable manner with        F, Cl, Br, I, C1-C18 alkyl, Silyl, OH, OR′, SH, SR′, SOR′,        SO₂R′, SO₃, NH₂, NHR′, or NR′₂, or an amino acid;    -   wherein R₃ and R₄ are independently H, OH, NH₂, or SH,        preferably OH;    -   wherein R₅ is either triphosphate, α-thiotriphosphate, or a salt        thereof;    -   wherein R′ is OH, alkyl, preferably C1-C6 alkyl or C2-C6        alkynyl;    -   wherein X is N, NH, or C;    -   wherein Y is O or NH₂;    -   wherein A, B, and E are independently C, N, O, S, P, or Se;    -   wherein n is 0 or 1; and    -   wherein M is H₂O or any metal, preferably a Group IA metal or a        Group IIA metal, more preferably Li⁺, Na⁺, K⁺, Mg²⁺, or Ca²⁺.

In another embodiment, the dye-labeled ribonucleotides of the inventioninclude purine-type compounds of the general formula VII:

-   -   wherein A is NH₂, OH, or O;    -   wherein R is H, O, NH₂, NHR′, S, CHR′, CR′₂, or halide,        preferably iodide, bromide, chloride, or fluoride, more        preferably chloride or fluoride;    -   wherein R′ is hydrogen or alkyl, preferably C1-C7 alkyl;    -   wherein R₁ and R₂ are independently H, OH, NH₂, or SH,        preferably OH;    -   wherein R₃ is either triphosphate, α-thiotriphosphate, or a salt        thereof;    -   wherein L is alkyl, preferably C1-C20 alkyl, C2-C20 alkenyl, or        C2-C20 alkynyl, or aryl substituted at from 0 to 3 positions in        a chemically reasonable manner with F, Cl, Br, I, C1-C18 alkyl,        Silyl, OH, OR′, SH, SR′, SOR′, SO₂R′, SO₃, or NR′₂;    -   wherein X is CR or N and Y is O, S, or NH; and    -   wherein the dye is any reporter group, preferably a        rhodamine-type dye, a fluorescein-type dye, an energy transfer        dye, or a cyanine dye.

The electrophoretic mobility and incorporation of dye labeledribonucleotides according to formula VII may be “tuned” by addingfunctional groups to the linker moiety between “X” and the dye.

The dye-labeled ribonucleotides of the invention also includepyrimidine-type compounds of the general formula VIII:

-   -   wherein X is N, NH, or C;    -   wherein Y is O or NH₂;    -   wherein R₁ and R₂ are independently H, OH, NH₂, or SH,        preferably OH;    -   wherein R₃ is either triphosphate, α-thiotriphosphate, or a salt        thereof;    -   wherein A is O, S, or NH;    -   wherein L is alkyl, preferably C1-C20 alkyl, C2-C20 alkenyl, or        C2-C20 alkynyl, or aryl substituted at from 0 to 3 positions in        a chemically reasonable manner with F, Cl, Br, I, C1-C18 alkyl,        Silyl, OH, OR′, SH, SR′, SOR′, SO₂R′, SO₃, or NR′₂;    -   wherein R′ is hydrogen or alkyl, preferably C1-C7 alkyl;    -   wherein n is 1 to 10; and    -   wherein the dye is any reporter group, preferably a        rhodamine-type dye, a fluorescein-type dye, an energy transfer        dye, or a cyanine-type dye.

The dye-labeled ribonucleotides of the invention also includepurine-type compounds of the general formula IX:

-   -   wherein R₄ is NH₂, OH, or O and R₅ is NH₂, OH, or H, provided        that if R₄ is NH₂, R₅ is H and if R₄ is O, R₅ is NH₂;    -   wherein R₁ and R₂ are independently H, OH, NH₂, or SH,        preferably OH;    -   wherein R₃ is either triphosphate, α-thiotriphosphate, or a salt        thereof;    -   wherein the dye is any reporter group, preferably a        rhodamine-type dye, a fluorescein-type dye, a big-dye, or        cyanine dyes, and    -   wherein R is a side chain for mobility tuning.

The dye-labeled ribonucleotides of the invention also includepyrimidine-type compounds of the general formula X:

-   -   wherein X is N, NH, or C;    -   wherein Y is O or NH₂;    -   wherein R₁ and R₂ are independently H, OH, NH₂, or SH,        preferably OH;    -   wherein R₃ is either triphosphate, α-thiotriphosphate, or a salt        thereof;    -   wherein the dye is any reporter group, preferably a        rhodamine-type dye, a fluorescein-type dye, a big-dye, or        cyanine dyes, and    -   wherein R is a side chain for mobility tuning.

Those of skill in the art will appreciate that many of the compoundsencompassed by the structures herein may exhibit the phenomena oftautomerism, conformational isomerism, geometric isomerism and/orstereoisomerism. As the formulae drawings within this specification andclaims can represent only one of the possible tautomeric, conformationalisomeric, enantiomeric or geometric isomeric forms, it should beunderstood that the invention encompasses any tautomeric, conformationalisomeric, enantiomeric and/or geometric isomeric forms of the compoundshaving one or more of the utilities described herein. As thenomenclature corresponds to the illustrated structural formulae, whichrepresent only one of several possible tautomeric forms (or resonancestructures) of the compounds, it will be understood that thesereferences are for convenience only, and that any such references arenot intended to limit the scope of the compounds described herein.

In addition, those of skill in the art also will recognize that thecompounds of the invention may exist in many different protonationstates, depending on, among other things, the pH of their environment.While the structural formulae provided herein depict the compounds inonly one of several possible protonation states, it will be understoodthat these structures are illustrative only, and that the invention isnot limited to any particular protonation state—any and all protonatedforms of the compounds are intended to fall within the scope of theinvention.

The compounds of the invention may bear multiple positive or negativecharges. Typically, the net charge of the labeled ribonucleotides of theinvention will be negative. The associated counter ions are typicallydictated by the synthesis and/or isolation methods by which thecompounds are obtained. Typical counter ions include, but are notlimited to, ammonium, sodium, potassium, lithium, halides, acetate,trifluoroacetate, etc., and mixtures thereof. It will be understood thatthe identity of any associated counter ion is not a critical feature ofthe invention, and that the invention encompasses the compounds inassociation with any type of counter ion. Moreover, as the compounds canexists in a variety of different forms, the invention is intended toencompass not only forms that are in association with counter ions(e.g., dry salts), but also forms that are not in association withcounter ions (e.g., aqueous or organic solutions).

The dye-labeled ribonucleotides of the invention are useful substratesfor DNA sequencing. As previously described, chain termination methodsgenerally require template-dependent primer extension in the presence ofchain-terminating nucleotides, resulting in a distribution of partialfragments which are subsequently separated by size. Standard dideoxysequencing methods utilize dideoxynucleoside triphosphates for chaintermination and a DNA polymerase such as the Klenow fragment of E. coliDNA polymerase I. See Sanger et al., supra.

Unlike the incorporation of a ddNTP, the incorporation of a dye-labeledribonucleotide according to the invention does not result in a chaintermination event. Rather, a DNA sequencing reaction comprising bothdye-labeled rNTPs and dNTPs produces a mixture of full-length primerextension products randomly substituted with rNTPs, which aresusceptible to cleavage at the 3′-5′ phosphodiester linkage between aribo- and an adjacent deoxyribonucleotide. Such primer extensionproducts provide an alternative to ddNTP-terminated extension productsfor the generation of sequence information. Following primer extensionin the presence of a single rNTP (e.g., rATP, rGTP, rCTP, or rUTP), thereaction mix can be treated with either alkali, heat, a ribonuclease orother means for hydrolyzing the extension products at each occurrence ofthe ribonucleotide to generate a series of extension products each witha dye-labeled 3′-ribonucleotide terminus. For a given target, analysisof the resulting sequencing products provides a sequencing ladder, i.e.,a series of identifiable signals in the G, A, T, and C lanescorresponding to the nucleic acid sequence of the target. The resultingsequencing ladder provides comparable information whether the methodutilizes ddNTPs or dye-labeled rNTPs.

When the DNA polymerase used in a sequencing reaction is a modifiedthermostable polymerase, such as those described in U.S. Pat. No.5,939,292, the dye-labeled ribonucleotides of the invention are usefulsubstrates for direct PCR sequencing (cycle sequencing). Because, incontrast to the incorporation of ddNTPs, the incorporation ofdye-labeled ribonucleotides does not cause chain termination, theinvention provides a method for the exponential amplification of targetDNA sequences by PCR. The invention, thus, provides greater sensitivityover currently available methods for cycle sequencing and allows thenucleotide sequence of a target to be determined from a small number oftemplate molecules.

DNA sequencing by PCR using the dye-labeled ribonucleotides of theinvention involves (i) annealing an oligonucleotide primer to atemplate; (ii) extending the primer with a DNA polymerase that canincorporate both dNTPs and rNTPs in a reaction comprising a mixture ofunlabeled dNTPs and at least one dye-labeled ribonucleotide of theinvention, (iii) treating the resulting primer extension products witheither alkali, heat, a ribonuclease, or other means for hydrolyzing theextension products at each occurrence of a ribonucleotide (each cleavageat the 3′-5′ phosphodiester linkage between a ribo- and an adjacentdeoxyribonucleotide results in a primer extension product that islabeled at the 3′-end), (iv) optionally, separating the resultingfragments that contain the primer from other fragments, (v) resolvingthe primer-containing extension products by means of, for example,high-resolution denaturing polyacrylamide/urea gel electrophoresis,capillary separation, or other resolving means; and (vi) detecting thefragments, for example, using a scanning spectrophotometer orfluorometer.

Methods for separating extension products that contain the primer fromother hydrolysis products will be apparent to those of skill in the art.Suitable methods include, but are not limited to, using biotinylatedprimers, which can be separated from other cleavage products using, forexample, avidin beads, or using hybridization based pull-out (HBP),wherein extension products containing the sequencing primers areseparated from other nucleic acids in the mixture using complementarypolynucleotides or oligonucleotides as described in U.S. Pat. No.6,124,092 to O'Neill et al., which is hereby incorporated herein byreference in its entirety. It will be apparent to the skilled artisanthat if primers complementary to both strands of a double-strandedtemplate are included in the reaction and those primers can beseparated, not only from the other cleavage products in the reaction,but also from each other, then the sequence of both strands of a DNAtemplate can be determined in one reaction.

It also will be apparent to the skilled artisan that using a set of fourribonucleotides of the invention each comprising a different dye labelenables one to determine the sequence of one or both strands of a DNAtemplate in a single reaction. The invention thus includes four colorsets of dye-labeled ribonucleotides that are characterized by equivalentelectrophoretic mobility and peak height evenness. The mobility of eachmember of such four color sets may be adjusted by, for example, thechoice between propargyl-ethyl-oxide-amino and propargylamino linkers,the choice between rigid and floppy linkers, and by includinghydrophobic or hydrophilic amino acid side chains in the linker. Anexemplary set of the dye-labeled ribonucleotides of the inventioncomprises rATP-PA-5-R6G, rCTP-PA-6-Rox, rUTP-PA-6-TAMRA, andrGTP-EO-5-R110.

The invention also encompasses methods for detecting mutations,including, but not limited to SNPs, in DNA by using pairs ofoligonucleotide primers selected to give a small amplicon containing amixture of dNTPs and dye-labeled rNTPs. In this aspect of the invention,two primers and a DNA polymerase that can incorporate both dNTPs andrNTPs are used to amplify a small segment of template DNA. The resultingamplicon is a chimeric DNA/dye-labeled RNA molecule, which can becleaved using alkali, heat, a ribonuclease, or other means forhydrolyzing the 3′-5′ phosphodiester linkage between a ribo- and anadjacent deoxyribonucleotide, leaving a single dye-labeledribonucleotide on the 5′ fragment. The resulting mixture is separatedby, for example, electrophoresis through a denaturing acrylamide ordialkylacrylamide matrix to give a specific microsequence in which thepresence or absence of the mutation can be unambiguously determined.

A general scheme for this method is illustrated in FIG. 1. In FIG. 1, asmall segment of genomic DNA is amplified using two primers of differentlength in the presence of dNTP's and a set of four dye-labeledribonucleotides according to the invention. In this example, Primer A is23 bp long and primer B is 21 bp long. The position of the mutation ofinterest is preferably, but does not have to be, the first nucleotidelocated 3′ to one, or both, of the primers. In this example, the SNP isthe first base 5′ to each primer. During the PCR reaction, dye-labeledribonucleotides, represented by the vertical ticks in the amplicon inFIG. 1, are incorporated along with the deoxynucleotides. Theincorporation of the dye-labeled ribonucleotides does not stop thepolymerization reaction, which can therefore go on to the otherextremity of the template. The strand produced, therefore, will containa priming site for the other primer and can be a template in thefollowing cycles of the PCR reaction.

The resulting amplicons are hydrolyzed, for example, in the presence ofNaOH to break them at every position where a dye-labeled ribonucleotidewas incorporated to produce a set of fragments each of which has one ofthe four ribonucleotides at its 3′ extremity. The extension productsfrom Primer A range from 24 to 44 bases in length. The extensionproducts from Primer B range from 22 to 44 bases in length. Theseproducts are separated on a denaturing matrix, in this example on a 16%acrylamide gel.

The electrophoresis results are illustrated in FIG. 2. Because Primer Cis shorter than Primer D, the smallest fragments result from theextension of Primer C. Because the SNP is located immediately adjacentto the 3′-end of Primer C, which is 24 nucleotides long, the smallestfragment detected, which is 25 nucleotides long, corresponds to the SNP.

FIG. 3 illustrates another embodiment of the invention, In this method,the microsequence (i.e., the partial sequence between the two primers)of both template strands is determined using primers of unequalmobility. In FIG. 3, one of the two primers, Primer F, is longer of thanthe other primer and contains a modified base “A” that does not permitextension in the 5′ direction, and that preferably, but not necessarily,is located midway along the primer sequence. In FIG. 3, Primer E, whichis 18 nucleotides long, and Primer F, which is 52 nucleotides long, areused to amplify a 5 bp portion of genomic DNA in the presence of dNTPsand a set of dye-labeled rNTPs according to the invention. After theresulting amplicons are cleaved using NaOH, labeled extension productsof Primer E will range from 19 to 45 nucleotides in length becausePrimer E cannot be extended beyond modified base A of Primer F. Thelabeled extension products of Primer F will range from 53 to 76nucleotides in length and do not overlap with the Primer E fragments.After the mixture of dye-labeled fragments is resolved byelectrophoresis through a denaturing matrix, the SNP which correspondsto the 21 nucleotide fragment of Primer E on the reverse strand and the55 nucleotide fragment of Primer F on the forward strand, can beunambiguously identified on both strands because the microsequences donot overlap. As alternatives to the use of a longer primer comprising amodified base, the method exemplified in FIG. 3 can also be practicedusing one primer comprising, for example, polyethylene glycol or aminoacids at its 5′-end.

In another embodiment of the invention, which is illustrated in FIG. 4,the extension products from each primer are separated prior to analysis,for example, using HBP, as shown, or biotinylated oligonucleotides,permitting the microsequence of both template strands to beunambiguously determined after electrophoresis in two lanes of adenaturing matrix.

The dye-labeled ribonucleotides of the invention have other uses, whichwill be apparent to one of skill in the art. For example, primerextension products comprising a mixture of dNTPs and dye-labeledribonucleotides may be cleaved by any of the methods described above toyield dye-labeled polynucleotide random fragments, which are useful ashybridization probes. The compounds of the invention also permit thesynthesis of dye-labeled RNAs, which are useful, for example, inquantifying the yield from an in vitro RNA synthesis and for preparingantisense and/or sense probes for in situ hybridization. The skilledartisan will appreciate additional uses for the dye-labeledribonucleotides of the invention from the examples below

Reference will now be made in detail to the exemplary embodiments of theinvention, examples of which are illustrated below.

Example 1

General Procedures: Sure Seal™ (Aldrich) or Puriss (Fluka) solvents wereused for reactions requiring anhydrous conditions. All reactions werecarried out under a positive argon or nitrogen atmosphere unlessotherwise noted. Reagents were added at room temperature unlessotherwise noted. Dyes obtained from manufacturing are activated asN-hydroxysuccinamide (NHS) esters and formulated to 5 mg per 60 μLdimethylsulfoxide (DMSO). Thin layer chromatography (TLC) was performedon silica gel GHLF₂₅₄250 μm plates (Analtech).

Analytical reverse phase (RP) high performance liquid chromatography(HPLC) was performed with a PE series 250 Binary LC pump interfaced witha PE 783A absorbance detector set at a wavelength λ 260 nm. For methodA, a Spheri-5 RP-18, 5 μm, 4.6×100 mm column was used with a lineargradient of 0-50% acetonitrile (AcCN) over 20 min, and a flow of 1mL/min with 0.1 M triethylammonium bicarbonate (TEAB) as buffer unlessotherwise noted.

For purification by ion exchange, HPLC was performed on a PE series 410B10 LC pump interfaced with a PE 785A absorbance detector (λ 260 nm) anda PE LC 240 fluorescence detector set at emission and absorbance valuesappropriate to the respective dye system. The column was developed witha linear gradient of 0-100% 0.1 M TEAB-1.5 M TEAB, 40% AcCN over 20 minat a flow rate of 1.5 mL/min. For two step purification methods ionexchange chromatography was followed by RP—C8 HPLC on a PE RP-8, 5 μm4.6×220 mm column with a linear gradient of 0-50% AcCN over 15 min at aflow rate of 1 mL/min with 0.1 M TEAB as the buffer unless otherwisenoted.

Preparatory reverse phase HPLC (Prep-HPLC) was performed on a WatersPrepLC 4000 system with a 40×450 mm RP-18 column using a linear gradientof 5-50% AcCN over 20 min, flow rate 50 mL/min with 0.1 M TEAAc bufferunless otherwise indicated (Method C)

Nuclear Magnetic Resonance spectra (NMR, ¹H, ³¹p, ¹⁹F, ¹³C) wererecorded on a Varian XL300 NMR spectrometer. Low resolution mass spectra(LRMS) were performed on a PE SCIEX AP-100 Electrospray Massspectrometer. Absorbance measurements were performed on a Perkin-ElmerLambda-Bio UV-Vis spectrophotometer.

Example 2

Synthesis of 7-Deaza-7-iodoadenosine.

Heterocycle preparation exemplified for6-chloro-7-iodo-pyrrolo[2,3-d]pyrimidine(6-chloro-7-deaza-7-iodopurine).See Seela, F et al., Helv. Chim. Acta, 73: 1602 (1990); Davol, J., J.Chem. Soc., 131 (1960); Wellcome Foundation, B. P. 812,366 (Apr. 22,1959).

The synthesis of 6-chloro-7-deaza-7-iodopurine 3.

Hilbert-Johnson Glycosylation Method.

Reference: Townsend et al., Nucleosides & Nucleotides 18: 153 (1999) andreferences therein.

Preparation of 10: N,O-Bis(trimethylsilyl)acetamide (BSA, 1.8 mL. 7.2mmol) was added to a stirring suspension of 3 (2 g, 7.3 mmol) in dryacetonitrile (AcCN, 50 mL) at room temperature. After stirring for 10min, 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose 9 (Aldrich, 3.6 g,7.2 mmol) was added, followed by the addition of trimethylsilyltrifluoromethanesulfonate (TMSOTf, 2.6 mL, 14.4 mmol) via syringe. Afterstirring for 10 min, the reaction mixture was placed in an oil bathpre-heated to 80° C. and stirred at 80° C. for an additional 1 h. Thereaction mixture was cooled to room temperature and then diluted withethylacetate (EtOAc, 200 mL) and saturated sodium bicarbonate (NaHCO₃,10 mL). The organic portion was washed successively with saturatedNaHCO₃ (1×50 mL), water (1×50 mL), and saturated sodium chloride (1×50mL), dried, and concentrated to give 5.7 g of crude 10 as a brown foam.The crude material was dissolved in EtOAc (40 mL) and filtered bysuction through a pre-moistened (EtOAc) plug of silica gel (150 g),rinsing with hexanes/EtOAc (4/1, 500 mL). The filtrate was concentratedto give 2.7 g of 10 (64%) as a yellow foam.

Preparation of 11: Ammonia gas was vigorously bubbled through asuspension of 10 (2.7 g, 3.7 mmol) in methanol (MeOH, 80 mL) at 0° C.over a period of 30 min. The reaction mixture was sealed in aglass-lined steel bomb and heated to 100° C. for 20 h. The reactionvessel was cooled to 0° C., opened, and concentrated to give 2.7 g of acrude dark brown oil. This material was recrystallized from water togive 0.58 g, 40% of 11 as a tan solid.

Example 3

Synthesis of 7-iodoguanosine (16):

Reference: Ramasamy, et al., J. Heterocyclic Chem. 25: 1893 (1988).

Preparation of 21: To a stirring solution of ribose sugar 24 [Kaskar etal., Synthesis, 1031 (1990)] (3.0 g, 10 mmol) dissolved in anhydrous THF(30 mL) and carbon tetrachloride(CCl₄, 1.2 mL, 12 mmol) cooled to −78°C. was added hexamethylphosphorus triamide (HMPT, 2.2 mL, 12 mmol). Theresulting solution was stirred at −78° C. until it had formed acolorless gel and then allowed to warm to room temperature over a periodof 1 h. The resulting solution of 22 was added to a stirring solution of23 (20 mmol) in CH₃CN (200 mL) at room temperature, and the reactionmixture was stirred for 17 h. The reaction mixture then was concentratedfollowed by dilution with water (100 mL). The aqueous solution wasextracted with ethyl acetate (2×100 mL). The combined organic portionswere washed with water (2×50 mL) and saturated NaCl (2×50 mL), driedover MgSO₄, and concentrated to give a dark brown oil. The crude oil wassuspended in EtOAc/hexanes (1:4, 100 mL) and the resulting precipitatewas filtered by suction washing with EtOAc/hexanes (1:4, 100 mL).Concentration of the filtrate afforded 4.8 g of a dark brown oil.Purification of this oil via column chromatography (silica gel, 500 g,EtOAc/hexanes, 1:4 as eluent) gave 1.85 g (41%) of 21 as a pale yellowsolid.

Preparation of 20: To a stirring solution of 21 (1.5 g, 3.2 mmol) inpyridine (20 mL) was added trimethylacetyl chloride (520 μL, 4.2 mmol)and the reaction mixture was stirred at room temperature for 17 h. Thereaction mixture was then taken up in 2:1. EtOAc/saturated sodiumbicarbonate (75 mL) and stirred for an additional 2 h. The organicportion was dried over MgSO4 and concentrated to give 1.74 g of 20 as abrown foam. Crude 20 was taken up in 9:1 hexanes/EtOAc (10 mL), filteredthrough a plug of silica gel (30 g, pre-moistened with 9:1hexanes/EtOAc), and rinsed with 9:1 hexanes/EtOAc (500 mL).Concentration of the filtrate gave 1.69 g (94%) of 20 as a colorlessfoam which was used without further purification in the next step.

Preparation of 19: To a stirring solution of 20 (1.6 g, 2.9 mmol) in DMF(15 mL) was added N-iodosuccinamide (NIS, 0.8 g, 3.2 mmol) and thereaction mixture was stirred in the dark for 17 h. ¹H NMR showed a 7:1mixture of 20:19 at which time a second equivalent of NIS (0.8 g) wasadded to the reaction mixture. After 4 h, the reaction was analyzed by¹H NMR to show a 1.6:1 mixture of 20:19. The reaction mixture was warmedto 45° C. and stirred for an additional 17 h. ¹H NMR showed no change inthe ratio of product to starting material from the previous analysis.The reaction was quenched by adding saturated sodium bicarbonate (25 mL)and extracted with EtOAc (200 mL). The organic portion was washed with5% sodium bisulfite (1×20 mL), water (2×25 mL), and saturated NaCl (2×25mL), dried over MgSO₄, and concentrated to dryness to give 1.25 g of 20and 19. This material again was dissolved in DMF (10 mL) followed by theaddition of NIS (0.8 g) and the reaction mixture was allowed to stir for17 h. ¹H NMR showed approximately 1:1 19:20. More NIS (0.8 g) was addedto the mixture, which was analyzed as before after 8 h showing 2.3:119:20. Another equivalent of NIS (0.8 g) was added and the reactionmixture was stirred overnight. ¹H NMR showed the reaction was complete.The reaction mixture was quenched, extracted, dried as before, andfiltered through a pre-moistened plug of silica gel (30 g, 4/1hexanes/EtOAc, rinsing with 4:1 hexanes/EtOAc (150 mL)). Concentrationafforded 1.29 g of 19 (67%) of suitable quality for the next step.

Preparation of 18: 19 (0.58 g, 0.87 mmol) was dissolved in freshlyprepared sodium methoxide (NaOMe, 1 M, 25 mL) and heated to reflux for 1h. The solution was cooled to 0° C. and the pH was adjusted to 6 withacetic acid (6 M). The solution was concentrated to dryness, suspendedin EtOAc/hexanes (1:1, 10 mL), and filtered through a plug of silica gel(30 g, rinsing with EtOAc/hexanes (1:1, 250 mL). Concentration of thefiltrate gave 0.32 g (64%) of 18 as a colorless foam of suitable qualityfor the next step.

Preparation of 17: An ice cold solution of trifluoroacetic acid/water(95:5, 10 mL) was added to 18 (0.32 g, 0.56 mmol) with stirring. Thesolution was stirred at 0° C. for 0.5 h. The reaction mixture wasconcentrated to dryness (co-evaporating with methanol (3×50 mL, toremove TFA)) and recrystallized with acetone/MeOH/DCM (5:2:4, 10 mL) togive 228 mg (97%) of 17 as a white powder.

Preparation of 16: A suspension of 17 (150 mg, 0.36 mmol) in NaOH (2 M,10 mL) was heated to reflux for 2 h. The solution was cooled to 0° C.and neutralized with acetic acid (AcOH, 2 M). 16 was collected byfiltration, washed with water, and dried to give 105 mg (72%) of 16 as atan solid.

Example 4 Preparation of Linkers

Synthesis of 3-Ethoxy-[2′-N-Trifluoroacetamido-]-1-Propyne (EO-Linker)28: NaH (60% dispersion in mineral oil, 22 g, 0.55 mol) was placed in a1 L RB flask, rinsed with dry THF (50 mL), and suspended in THF (150mL). The suspension was cooled to 0° C., followed by the dropwiseaddition of 2-aminoethanol (32 mL, 0.5 mol) with stirring over a periodof 0.5 h. The reaction mixture was diluted with additional THF (500 mL)and allowed to warm to room temperature over a period of 2 h. Theresulting solution was cooled to 0° C., followed by the slow addition ofpropargyl bromide (45 mL, 0.5 mol) over a period of 0.5-1 h. Thereaction mixture was allowed to gradually warm to room temperature andstirred for 17 h. The reaction mixture was filtered by suction, rinsingwith THF (100 mL), concentrated to a black syrup, and distilled byvacuum (6 mmHg, 35-42° C.) to give 17 g of desired amine 28′. 28′ wasadded to ethyltrifluoroacetate (30 g, 0.21 mol) at 0° C. over a periodof 0.5 h, followed by stirring at room temperature for 2 h. The reactionmixture was concentrated and distilled by vacuum (92-93° C., 6-7 mm Hg).This was repeated twice to give 15 g of 28 of suitable purity for thenext step.

Synthesis of N-3-trifluoroacetamido-1-propyne (PA-Linker) 27. See Hobbs,Jr. et al., U.S. Pat. Nos. 5,047,519 and 5,151,507.

Example 5

Linker Coupling: See Hobbs, Jr. et al., U.S. Pat. No. 5,047,519.

Ribo-Adenosine PA, EO-Linker Coupling: Preparation of 1a and 1e

Preparation of 1a: To a stirring solution of 11 (100 mg, 0.25 mmol) inN,N-dimethylformamide (DMF, 4 mL) was added in the following orderN-3-trifluoroacetamido-1-propyne 27 (PA-linker, 190 μL, 1.25 mmol),triethylamine, (TEA, 110 μL, 0.76 mmol), cuprous iodide (Cul, 10 mg,0.05 mmol), and tetrakis(triphenylphosphine)palladium (30 mg, 0.025mmol). The reaction mixture was stirred for 17 h and then quenched bythe addition of Dowex-1 carbonate form (3 g) and rinsed withdichloromethane(DCM)/MeOH (1:1, 200 mL). The filtrate was concentratedand the crude product was purified by column chromatography [silica gel,50 g, eluting with DCM/MeOH (9:1, 200 mL) followed by DCM/MeOH (8:2, 200mL)] to give 84 mg of 2 as a brown solid.

Preparation of 1e: To a stirring solution of 11 (100 mg, 0.26 mmol) inDMF (1 mL) was added in the following order3-ethyloxy-[2′-N-trifluoroacetamido-]-1-propyne 28 (EO-linker, 140 μL,0.76 mmol), TEA (110 μL, 0.76 mmol), Cul (10 mg, 0.05 mmol), andtetrakis(triphenylphosphine)palladium (30 mg, 0.025 mmol). The reactionmixture was allowed to stir for 17 h followed by quenching with Dowex-1carbonate form (3 g). The reaction mixture was filtered over a plug ofcelite (10 g) and rinsed with DCM/MeOH (1:1, 100 mL). The filtrate wasconcentrated and the crude product was purified by column chromatography[silica gel, 50 g, eluting with DCM/MeOH (9:1, 200 mL)] to give 90 mg of1e (71%) as a brown solid.

Preparation of 2a and 2e: See Hobbs, Jr. et al., U.S. Pat. Nos.5,047,519 and 5,151,507.

Preparation of 2a: To a solution of 16 (50 mg, 0.12 mmol) in dry DMF (2mL) at room temperature was added 27 (100 μL, 0.61 mmol), cuprous iodide(Cul, 8 mg, 0.04 mmol), tetrakis(triphenylphosphine)palladium(0) (14 mg,0.012 mmol), and Et₃N (50 μL, 0.37 mmol). The reaction mixture wasstirred in the dark for 17 h, followed by the addition of ion exchangeresin (strongly basic, Dowex 1×50 carbonate form, 3 g). The resultingmixture was stirred for 0.5 h, filtered though a plug of celite (10 g,rinsing with DCM/MeOH, 1:1, 100 mL), and concentrated to give a darkbrown oil. Purification via column chromatography (silica gel, 40 g,DCM/MeOH 85:15 as eluent) gave 40 mg (75%) of 2a as a tan solid.Alternatively, the triethylammonium salt may be removed without addingion exchange resin by careful column chromatography.

Preparation of 2e: To a solution of 16 (100 mg, 0.24 mmol) in dry DMF (2mL) at room temperature was added 28 (130 μL, 0.74 mmol), cuprous iodide(Cul, 9 mg, 0.05 mmol), tetrakis(triphenylphosphine)palladium(0) (57 mg,0.05 mmol), and Et₃N (100 μL, 0.74 mmol). The reaction mixture wasstirred in the dark for 17 h and concentrated to give a dark brown oil.Purification via column chromatography (silica gel, 40 g, DCM/MeOH 95:5,90/10, 85/15 200 mL each, as eluent) gave 60 mg (53%) of 2e as a tansolid.

Preparation of rC-PA-NHTFA (3a): To a stirring solution of 15 (100 mg,0.27 mmol) in DMF (4 mL) was added in the following orderN-3-trifluoroacetamido1-propyne 27 (PA-linker, 200 μL, 1.35 mmol),triethylamine, (TEA, 115 μL, 0.81 mmol), cuprous iodide (Cul, 15 mg,0.08 mmol), and tetrakis(triphenylphosphine)palladium (35 mg, 0.027mmol). The reaction mixture was stirred for 72 h, followed by theaddition of Dowex-1 carbonate form (3 g). The quenched reaction mixturewas filtered over a plug of celite (10 g), and rinsed with DCM/MeOH(1:1, 200 mL). The filtrate was concentrated and the crude product waspurified by column chromatography [silica gel, 50 g, eluting withDCM/MeOH (9:1, 200 mL) followed by DCM/MeOH (8:2, 200 mL)] to give 60 mgof 3a (57%) as a brown solid.

Preparation of rC-EO-NHTFA (3e): To a stirring solution of 15 (100 mg,0.26 mmol) in DMF (1 mL) was added in the following order3-ethyloxy-[2′-N-trifluoroacetamido-]-1-propyne 28 (EO-linker, 140 μL,0.76 mmol), TEA (110 μL, 0.76 mmol), Cul (10 mg, 0.05 mmol), andtetrakis(triphenylphosphine)palladium (30 mg, 0.027 mmol). The reactionmixture was stirred for 23 h, followed by quenching with Dowex-1carbonate form (3 g). The quenched reaction mixture filtered over a plugof celite (10 g) and rinsed with DCM/MeOH (1:1, 100 mL). The filtratewas concentrated and the crude product was purified by columnchromatography [silica gel, 50 g, eluting with DCM/MeOH (85:15, 200 mL),then DCM/MeOH (80:20, 100 mL)] to give a mixture of 3e/15 (8:1) as abrown solid mixture.

Preparation of rC-PA-NHTFA (3a) using 5-Bromocytidine (15b): To astirring solution of 15b (250 mg, 0.78 mmol) in DMF (5 mL) was added inthe following order N-3-trifluoroacetamido-1-propyne 27 (PA-linker, 320μL, 2.33 mmol), triethylamine, (TEA, 320 μL, 2.33 mmol), cuprous iodide(Cul, 30 mg, 0.16 mmol), and tetrakis(triphenylphosphine)palladium (540mg, 0.47 mmol). The reaction mixture was placed in a pre-heated oil bathat 80° C. and stirred for 4 h. The reaction mixture was concentrated,diluted with DCM/MeOH (80/20, 5 mL), and purified via columnchromatography (silica gel, 50 g, 9/1 DCM/MeOH, 200 mL, then DCM/MeOH80/20) to give 150 mg of 3a (50%) as a brown solid (90% HPLC purity).

Preparation of r-C-EO-NHTFA (3e) using 5-Bromocytidine (15b): To astirring solution of 15b (100 mg, 0.31 mmol) in dry DMF (2 mL) was added3-ethyloxy-[2′-N-trifluoroacetamido-]-1-propyne 28 (EO-linker, 170 μL,0.93 mmol), Cul (15 mg, 0.079 mmol),tetrakis(triphenylphosphine)palladium (150 mg, 0.13 mmol)[2], and TEA(110 μL, 0.76 mmol) at room temperature. The reaction mixture was placedin a pre-heated oil bath at 80° C. and stirred for 1.5 h. The reactionmixture was cooled to room temperature followed by the addition ofDowex-1 carbonate form (3 g) and allowed to stir for an additional 0.5h. The solution was filtered over a plug of celite (10 g) and rinsedwith DCM/MeOH (1:1, 100 mL). The filtrate was concentrated and purifiedby column chromatography [silica gel, 50 g, eluting with DCM/MeOH(90:10, 200 mL), then DCM/MeOH (80:20, 100 mL)] to give 70 mg of 3e(52%).

Example 6

TRIPHOSPHATE SYNTHESIS General Procedure.

Reference: Ludwig, J. Acta Biochim. et Biophys. Acad. Sci. Hung., 16:131(1981).

In Scheme VI, the nucleobase in compounds 1a, 1b, and 1c is adenosine;the nucleobase in compounds 2a, 2b, and 2c is guanosine; the nucleobasein compounds 3a, 3b, and 3c is cytosine; and the nucleobase in compounds4a, 4b, and 4c is uridine.

To a stirring solution cooled to −10° C. of nucleoside (0.2 mmol) in drytrimethylphosphate (TMP; 0.5 mL) was added phosphorous oxychloride(POCl₃; 0.44 mmol, 2.2 eq.) via syringe. To monitor the progress of thereaction, 5 μL aliquots were taken from the reaction mixture, hydrolyzedin 0.2 mL of 0.1 M TEAB buffer, and 20 μL samples were analyzed byRP-HPLC using method A. The reaction was normally complete within 2-4 h,although, in some cases, longer reaction times were needed. In certaincases, additional POCl₃ was needed for the reaction to be completed(i.e., up to 6-8 equivalents). Additional POCl₃ did not effect the siteof phosphate addition. After the reaction was complete, the reaction wasquenched by the addition of a vigorously stirred solution ofbis-tri-n-butylammonium pyrophosphate (TBAPP; 6 eq, 1.2 mmol), 0.5 M indry dimethylformamide (DMF) (2.0 mL), and tri-n-butylamine (TBA; 10 eq,2 mmol, 0.5 mL). The resulting reaction mixture was stirred for 1 minand then poured into a TEAB solution (1.0 M, 20 mL) at 0° C. Theresulting solution was allowed to stand for a period of 3 h,concentrated, and purified via preparatory HPLC using method C. Theresulting triphosphate was concentrated, taken up in 0.1 M TEAB (10-20mL), and stored in the freezer at −20° C.

Preparation of rATP-PA-NHTFA (1c): To a stirring solution of anhydrous1a (50 mg, 0.12 mmol) in dry TMP (1 mL) at −10° C. was added POCl₃(99.999% Aldrich, 25 mL, 0.26 mmol). The reaction was monitored by HPLC(Method A, 5 μL aliquots in 100 μL 1 M TEAB). The retention time (RT) of1a=11.7 min, the RT of 1b=9.1 min. An aliquot withdrawn from thereaction at 0.5 h showed a ratio of 47/41 1a/1b. An aliquot withdrawn at1.5 h showed a ratio of 41/46 1a/1b. An aliquot withdrawn at 2.5 hshowed a ratio of 33/64 1a/1b. An aliquot withdrawn at 7 h showed aratio of 19/70 1a/1b. After 7 hours, the reaction mixture was tightlysealed, placed in the freezer at −20° C., and allowed to stand for 48 h.An aliquot of the reaction showed a ratio of 13/69 1a/1b. The reactionmixture was quenched by the addition of a vigorously stirred solution ofTBAPP (330 mg, 0.72 mmol) in TBA (290 mL, 1.2 mmol) in DMF (1.0 mL) at0° C. with stirring over a period of 1 min. This was followed by pouringthe mixture into a solution of TEAB (1 M, 10 mL) at 0° C. The crudetriphosphate 1c then was allowed to stand over a period of 1 h. Thereaction mixture was concentrated to 4 mL and purified twice usingpreparatory HPLC (Method C,) to give 1c of suitable purity for the nextstep.

Preparation of rATP-EO-NHTFA (1g): To a stirring solution of 1e (90 mg,0.20 mmol) prepared as in Example 6 in dry TMP (1.5 mL) at −10° C. wasadded POCl₃ (99.999% Aldrich, 40 μL, 0.43 mmol) from a freshly openedampoule. The reaction was monitored by HPLC (Method A, 5 μL aliquots in100 μL 1 M TEAB). The retention time (RT) of 1e=11.4 min. The RT of 1f(monophosphate)=8.6 min. An aliquot withdrawn from the reaction at 0.5 hshowed a ratio of 1/1 1e/1f. An aliquot withdrawn at 1 h, 50 min showeda ratio of 1/3 1e/1f. After 4 h, a second 2.2 equivalents of POCl₃ (40μL, 0.43 mmol) were added. The reaction mixture was tightly sealed,placed in the freezer at −20° C., and allowed to stand for 17 h. Analiquot of the reaction showed quantitative conversion to themonophosphate 1f. The reaction mixture was quenched by the addition of avigorously stirred solution of TBAPP (900 mg, 2.0 mmol) in TBA (660 μL,2.8 mmol) in DMF (2.0 mL) at 0° C. with stirring over a period of 1 min.This was followed by pouring the mixture into a solution of TEAB (0.1 M,25 mL) at 0° C. and the crude triphosphate 1g was allowed to stand for 3h. The reaction mixture was concentrated to 3 mL, filtered using a 0.2micron in-line filter, and purified by preparatory HPLC (Method C) togive 1 g of suitable purity for the next step.

Preparation of rGTP-PA-NHTFA (2c): To a stirring solution of 2a (38 mg,0.09 mmol) prepared as in Example 6 in dry TMP (1.0 mL) at −10° C. wasadded POCl₃ (99.999% Aldrich, 18 μL, 0.19 mmol) from a freshly openedampoule. The reaction was monitored by HPLC (Method A, 5 μL aliquots in100 μL 1 M TEAB). The retention time (RT) of 2a=10.4 min. The RT of 2b(5′-O-monophosphate)=8.1 min. An aliquot withdrawn from the reaction at0.33 h showed a ratio of 19/1 2a/2b. An aliquot withdrawn at 1 h, 50 minshowed a ratio of 9/1 2a/2b. The reaction mixture was tightly sealed,placed in the freezer at −20° C., and allowed to stand for 17 h. HPLCanalysis showed a ratio of 3/1 2a/2b. After 2 h, an additional 1.5equivalents of POCl₃ (12 μL, 0.13 mmol) was added with stirring at 0° C.HPLC analysis of the reaction after a further 3 h, 40 min showed a ratioof 1/1 2a/2b. An additional 1.5 equivalents of POCl₃ (12 μL, 0.13 mmol;5.2 equivalents total) was added while maintaining the reactiontemperature between 4 and −10° C. The reaction mixture was tightlysealed, placed in the freezer at −20° C., and allowed to stand for 18 h.HPLC analysis of the reaction after this period showed a ratio of 0.8/12a/2b. Addition of a third 1.5 eq of POCl₃ (12 μL, 0.13 mmol) was madewith stirring at 0° C. over a period of 2.25 h. The reaction mixtureagain was sealed and placed in the freezer at −20° C. for 48 h. HPLCshowed a ratio of 78/12 2b/2a. The reaction mixture was quenched by theaddition of a vigorously stirred solution of TBAPP (720 mg, 1.6 mmol) inTBA (630 μL, 2.7 mmol) in DMF (2.0 mL) at 0° C. with stirring over aperiod of 1 min. This was followed by pouring the mixture into asolution of TEAB (0.1 M, 25 mL) at 0° C. and the crude triphosphate 2cwas allowed to stand for 3 h. The reaction mixture was concentrated to3-4 mL, filtered using a 0.2 micron in-line filter, and purified usingpreparatory HPLC (Method C). This purification was repeated to give 2cof suitable purity for the next step. ³¹P NMR showed a 10/1 ratio of2c/2b.

Preparation of rGTP-EO-NHTFA (2g): To a stirring solution of 2e (33 mg,0.07 mmol) prepared as in Example 6 in dry TMP (1.0 mL) at −10° C. wasadded POCl₃ (99.999% Aldrich, 26 μL, 0.28 mmol, 4 eq.). Another 4equivalents of POCl₃ (99.999% Aldrich, 26 μL, 0.28 mmol) were addedafter 1.5 h, while the reaction temperature was mained between 4 and−10° C. The reaction mixture was tightly sealed, placed in the freezerat −20° C. and allowed to stand for 24 h. HPLC (Method A, 5 μL aliquotsin 100 μL 1 M TEAB) showed that the starting material 2e (RT=10.2 min)was consumed with the appearance of the 5′-O-monophosphate 2f (RT=7.8min). The reaction mixture was quenched by the addition of a vigorouslystirred solution of TBAPP (280 mg, 0.62 mmol) in TBA (215 μL, 0.9 mmol)in DMF (1.0 mL) at 0° C. with stirring over a period of 1 min. This wasfollowed by pouring the mixture into a solution of TEAB (0.1 M, 20 mL)at 0° C. and the crude triphosphate 2g was allowed to stand for 3 h. Thereaction mixture was concentrated to 3-4 mL, filtered using a 0.2 micronin-line filter, and purified via preparatory HPLC (Method C) followed byflash ion exchange chromatography (DEAE Sephadex, 25A, 2 g) using astepwise gradient of 0.1 M TEAB (100 mL), 0.25 M TEAB (100 mL), 0.3 MTEAB (50 mL), 0.35 M TEAB (50 mL), 0.38 M TEAB (50 mL), 0.42 M TEAB (50mL), 0.45 M TEAB (50 mL), 0.5 M TEAB (50 mL), 0.6 M TEAB (50 mL), 0.7 MTEAB (50 mL), 0.8 M TEAB (50 mL), 1.0 M TEAB (50 mL). Fractions (0.42 M−1.0 M TEAB, analysis by ³¹P NMR) were pooled and concentrated to give 3mL of a 5.4 mM solution of triphosphate 2g of suitable purity for thenext step.

Preparation of rCTP-PA-NHTFA (3c): To a stirring solution of 3a (50 mg,0.13 mmol) prepared as in Example 6 in dry TMP (1 mL) at −10° C. wasadded POCl₃ (99.999% Aldrich, 26 μL, 0.28 mmol). The reaction wasmonitored by HPLC (Method A, 5 μL aliquots in 100 μL 1 M TEAB). Theretention time (RT) of 3a=9.6 min. The RT of 3b=7.6 min. At 0.5 h, theratio of 3a/3b was 3/1. An aliquot withdrawn at 1.5 h showed a 3a/3bratio of 45/40. An aliquot withdrawn at 5 h showed a 3a/3b ratio of20/61. The reaction mixture was tightly sealed, placed in the freezer at−20° C., and allowed to stand for 48 h. An aliquot of the reactionmixture showed mostly 3b. The reaction mixture was quenched by theaddition of a vigorously stirred solution of TBAPP (350 mg, 0.76 mmol)in TBA (300 μL, 1.3 mmol) in DMF (1.0 mL) at 0° C. with stirring over aperiod of 1 min. This was followed by pouring the mixture into asolution of TEAB (0.1 M, 25 mL) at 0° C. and the crude triphosphate 3cwas allowed to stand for 3 h. The reaction mixture was concentrated to 4mL and purified using preparatory HPLC (Method C) to give 3c of suitablepurity for the next step.

Preparation of rCTP-EO-NHTFA (3g): To a stirring solution of 3e (70 mg,0.16 mmol) prepared as in Example 6 in dry TMP (1.0 mL) at −10° C. wasadded POCl₃ (99.999% Aldrich, 33 μL, 0.35 mmol) from a freshly openedampoule. The reaction was monitored by HPLC (Method A, 5 μL aliquots in100 μL 1 M TEAB). The RT of 3e=9.4 min. The RT of 3f (monophosphate)=7.3min. At 0.5 h, the ratio of 3f/3e was 3/1. The reaction mixture wastightly sealed and placed in the freezer at −20° C. and allowed to standfor 17 h. An aliquot of the reaction showed a 3f/3e ratio of 4/1. Asecond portion of POCl₃ (99.999% Aldrich, 33 μL, 0.32 mmol, 2.2 eq) wasadded at a temperature between 0° C. and −10° C. and the reactionmixture was stirred for 15 min. The reaction mixture then was quenchedby the addition of a vigorously stirred solution of TBAPP (500 mg, 1.1mmol) in TBA (400 μL, 1.7 mmol) in DMF (1.0 mL) at 0° C. with stirringover a period of 1 min. This was followed by pouring the mixture into asolution of TEAB (0.1 M, 25 mL) at 0° C. and the crude triphosphate 3gwas allowed to stand for 2-3 h. The reaction mixture was concentrated to3 mL, filtered using a 0.2 micron in-line filter, and purified usingpreparatory HPLC Method C) to give 3g of suitable purity for the nextstep.

Preparation of rUTP-PA-NHTFA (4c): To a stirring solution of 4a(prepared from 5-iodouracil as exemplied for 3a, 50 mg, 0.13 mmol) indry TM P (0.5 mL) at −10° C. was added POCl₃ (99.999% Aldrich, 26 μL,0.28 mmol). The reaction was monitored by HPLC (Method A, 5 μL aliquotsin 100 μL 1 M TEAB). The RT of 4a RT=8.8 min. The RT of 4b=6.8 min. At0.75 h, the ratio of 4a/4b was 9/1. An aliquot withdrawn at 5 h showed a4a/4b ratio of 45/55. The reaction mixture was tightly sealed and placedin the freezer at 4° C. and allowed to stand for 18 h. An aliquot of thereaction mixture showed mostly 4b. The reaction mixture was quenched bythe addition of a vigorously stirred solution of TBAPP (290 mg, 0.64mmol) in TBA (210 μL, 0.89 mmol) in DMF (1.0 mL) at 0° C. with stirringover a period of 1 min. This was followed by pouring the mixture into asolution of TEAB (0.1 M, 40 mL) at 0° C. and the crude triphosphate 4cwas allowed to stand for 3 h. The reaction mixture was concentrated to 4mL and purified using preparatory HPLC (Waters, Delta Prep, Method C,Appendix II) to give 4c of suitable purity for the next step.

Preparation of rUTP-EO-NHTFA (4g): To a stirring solution of 4e(prepared from 5-iodouracil as exemplied for 3e, 50 mg, 0.11 mmol) indry TMP (0.5 mL) at −5° C. was added POCl₃ (99.999% Aldrich, 33 μL, 0.35mmol) from a freshly opened ampoule. The reaction mixture was tightlysealed, placed in the freezer at −20° C., and allowed to stand for 17 h.The reaction was monitored by HPLC (Method A, 5 μL aliquots in 100 μL 1M TEAB). An aliquot of the reaction showed 14/1 4e/4f (RT of 4e=8.9 min,RT of 4f (monophosphate)=7.4 min). After stirring for 0.5 h, a secondportion of POCl₃ (99.999% Aldrich, 33 μL, 0.32 mmol, 2.2 eq)) was addedat a temperature between 0° C. and −10° C. and the reaction mixture wasallowed to stir for 10 min. An aliquot of the reaction mixture showed a4e/4f ratio of 5/1. After stirring for an additional 5 h, a thirdportion of POCl₃ (99.999% Aldrich, 33 μL, 0.32 mmol, 2.2 eq)) was addedat a temperature between 0° C. and −10° C. and the reaction mixture wasstirred for 0.5 h. An aliquot of the reaction mixture showed a 4e/4fratio of 1/1. The reaction mixture was tightly sealed, placed in thefreezer at −20° C., and allowed to stand for 17 h. The reaction mixturewas removed from the freezer and stirred at 0° C. for 1 h, 15 minfollowed by the addition of a vigorously stirred solution of TBAPP (720mg, 1.6 mmol) in TBA (540 μL, 2.3 mmol) in DMF (2.0 mL) at 0° C. Afterstirring for 2 min, the reaction mixture was poured into TEAB (0.1 M, 25mL) at 0° C. and the crude triphosphate 4g was allowed to stand for 2-3h. The reaction mixture was concentrated to 3 mL, filtered using a 0.2micron in-line filter, and purified using preparatory HPLC (Method C) togive 4g of suitable purity for the next step.

Example 7

Trifluoroacetamide Deprotection and Nucleotide Formulation: GeneralProcedure.

The nucleoside triphosphate in 0.1 M TEAB buffer was concentratedessentially to dryness via rotary evaporation. The resulting sample wasdiluted with ammonium hydroxide (5-10 mL), the solution was allowed tostir at room temperature for the appropriate period of time (0.5 to 3 h)and then concentrated. The remaining NH₄OH was removed viaco-evaporation with 0.1 M TEAB (2×10-20 mL). The deprotected nucleotidewas diluted with 0.1 M TEAB (3-5 mL) and the concentration and yieldwere determined according to Beer's Law (Equation 1) using the followingextinction coefficients for the deoxyribose nucleosides: dATP=15,200,dCTP=9,300, dGTP=13,700, dUTP=9,600.C _(mM)=[(A·Vol _(tot) /Vol _(sample))/(ε_(nucleoside cm) ⁻¹ _(M) ⁻¹·b)]×1000  Equation 1

Where C_(mM) is the concentration (millimolar, mM), A is the observedabsorbance, Vol_(tot) and Vol_(sample), are the total and samplevolumes, respectively, b is the path length, and ε is the molarextinction coefficient.

Preparation of rATP-PA-NH₂ (1d): A solution of 1c in 0.1 M TEAB (˜10 mL)was concentrated followed by dilution with concentrated NH₄OH (5 mL).The reaction mixture was stirred for 40 min and concentrated using arotary evaporator. The remaining NH₄OH was removed by diluting theresidue with 0.1 M TEAB (5 mL) and re-concentrating. This was repeated 2times. Formulation in 0.1 M TEAB gave a 12.09 mM solution of 1d (λ_(max)280 nm, ˜6 mL, 60 μmol, 50%, from 1a).

Preparation of rATP-EO-NH₂ (1h): A solution of 1g in 0.1 M TEAB (˜10 mL)was concentrated followed by dilution with concentrated NH₄OH (6 mL).The reaction mixture was stirred for 3 h and concentrated using a rotaryevaporator. The remaining NH₄OH was removed by diluting the residue with0.1 M TEAB (5 mL) and re-concentrating. This was repeated 2 times.Formulation in 0.1 M TEAB gave a 17.7 mM solution of 1h (λ_(max) 280 nm,˜7 mL, 124 μmol, 62% from 1e).

Preparation of rGTP-PA-NH₂ (2d): A solution of 2c in 0.1 M TEAB (˜10 mL)was concentrated followed by dilution with concentrated NH₄OH (7 mL).The reaction mixture was stirred for 30 min and concentrated using arotary evaporator. The remaining NH₄OH was removed by diluting theresidue with 0.1 M TEAB (5 mL) and re-concentrating. This was repeated 2times. Formulation in 0.1 M TEAB gave ˜3 mL of a 7.96 mM solution of 2d(λ_(max) 234 nm, 274 nm, 292 nm); Low Resolution MS, M-1, 574), 24 μmol,27%, from 2a).

Preparation of rGTP-EO-NH₂ (2h): A solution of 2g in 0.1 M TEAB (˜10 mL)was concentrated followed by dilution with concentrated NH₄OH (7 mL).The reaction mixture was stirred for 30 min and concentrated using arotary evaporator. The remaining NH₄OH was removed by diluting theresidue with 0.1 M TEAB (5 mL) and re-concentrating. This was repeated 2times. Formulation in 0.1 M TEAB gave ˜5 mL of a 5.37 mM solution of 2h(λ_(max) 238 nm, 274 nm, 292 nm); Low Resolution MS, M-1, 618,RT_(method A)=5.2 min), 27 μmol, 26%, from 2e).

Preparation of rCTP-PA-NH₂ (3d): A solution of 3c in 0.1 M TEAB (˜10 mL)was concentrated followed by dilution with concentrated NH₄OH (5 mL).The reaction mixture was stirred for 2.5 h and concentrated by rotaryevaporation. The remaining NH₄OH was removed by diluting the residuewith 0.1 M TEAB (5 mL) and re-concentrating. This was repeated 2 times.Formulation in 0.1 M TEAB gave 3 mL of a 9.7 mM solution of 3d (λ_(max)238 nm, 296 nm; 29 μmol, 23%, from 3a).

Preparation of rCTP-EO-NH₂ (3h): A solution of 3g in 0.1 M TEAB (˜10 mL)was concentrated followed by dilution with concentrated NH₄OH (5 mL).The reaction mixture was stirred for 3 h and concentrated by rotaryevaporation. The remaining NH₄OH was removed by diluting the residuewith 0.1 M TEAB (5 mL) and re-concentrating. This was repeated 2 times.Formulation in 0.1 M TEAB gave 4.5 mL of a 13.5 mM solution of 3h(λ_(max) 296 nm, 61 μmol, 38%, from 3e).

Preparation of rUTP-PA-NH₂ (4d): A solution of 4c in 0.1 M TEAB (˜10 mL)was concentrated followed by dilution with concentrated NH₄OH (5 mL).The reaction mixture was stirred for 4 h and concentrated by rotaryevaporation. The remaining NH₄OH was removed by diluting the residuewith 0.1 M TEAB (5 mL) and re-concentrating. This was repeated 2 times.Formulation in 0.1 M TEAB gave 1 mL of a 13.5 mM solution of 4d (λ_(max)234 nm, 288 nm, 14 μmol, 11%, from 4a).

Preparation of rUTP-EO-NH₂ (4h): A solution of 4g in 0.1 M TEAB (˜10 mL)was concentrated followed by dilution with concentrated NH₄OH (5 mL).The reaction mixture was stirred for 3 h and concentrated by rotaryevaporation. The remaining NH₄OH was removed by diluting the residuewith 0.1 M TEAB (5 mL) and re-concentrating. This was repeated 2 times.Formulation in 0.1 M TEAB gave 3 mL of a 6.5 mM solution of 4h (λ_(max)232 nm, 292 nm, 20 μmol, 17%, from 4e).

Example 8

Dye-Nucleoside Coupling: General Procedure.

Approximately 0.3 μmol of ribonucleotide triphosphate in 0.1 M TEAB wasconcentrated to dryness with a centrifuge-vacuum apparatus (e.g., aSpeed-Vac). The resulting solid was dissolved in sodium bicarbonate (250mM, 50 μL) adjusted to pH 9 with 1.0 M NaOH followed by the addition ofthe dye-NHS ester (0.8 μmol, 5 μL, conc.=5 mg/60 μL DMSO, 2.7 eq),mixed, and allowed to stand for 17 h. The reaction mixture was brieflycentrifuged to remove particulates and the reaction mixture was purifiedvia ion exchange HPLC followed by RP—C8 HPLC according to the proceduresdescribed in Example 1. When TEAA buffer was used in the RP—C8purification step, TEAB was added to prevent acid build-up duringconcentration. After RP—C8 purification, the dye-labeled ribonucleotidetriphosphate was formulated in3-[cyclohexylamino]-2-hydroxy-1-propanesulfonic acid (CAPSO, 50 mM,50-100 μL). A 5 μL portion was diluted with 0.1 M TEAB (500 μL) and anUV spectrum was obtained. The concentration (mM) was calculated fromEquation 2.C _(mM)=[(A·Vol _(tot) /Vol _(sample))/(ε_(dye cm) ⁻¹ M ⁻¹·b)]×1000  Equation 2where ε_(dye cm) ⁻¹ is the molar extinction of the dye. Solutions ofdye-labeled ribonucleotide analogs were prepared at the followingconcentrations:

rATP-PA-5-R6G (1i): 505 μM in 95 μL of 50 mM CAPSO.

rATP-PA-d-R6G-2 (1j): 104 μM in 25 μL of 50 mM CAPSO.

rATP-EO-5-R6G (1k): 758 μM in 95 μL of 50 mM CAPSO.

rATP-EO-d-R6G-2 (1l): 34 μM in 95 μL of 50 mM CAPSO.

rGTP-PA-5-R110 (1i): 63 μM in 25 μL of 50 mM CAPSO.

rGTP-PA-d-R110-2 (2j): 664 μM in 45 μL of 50 mM CAPSO.

rGTP-EO-5-R110 (2k): 123 μM in 25 μL of 50 mM CAPSO.

rGTP-EO-d-R110-2 (2l): 173 μM in 55 μL of 50 mM CAPSO.

rCTP-PA-6-ROX (3i): 162 μM in 115 μL of 50 mM CAPSO.

rCTP-PA-d-ROX-2 (3i): 121 μM in 55 μL of 50 mM CAPSO.

rCTP-EO-6-ROX (3k): 71 μM in 95 μL of 50 mM CAPSO.

rCTP-EO-d-ROX-2 (3l): 172 μM in 55 μL of 50 mM CAPSO.

rUTP-PA-6-TAMRA (4i): 684 μM in 95 μL of 50 mM CAPSO.

rUTP-PA-d-TAMRA-2 (4j): 818 μM in 95 μL of 50 mM CAPSO.

rUTP-EO-6-TAMRA (4k): 404 μM in 95 μL of 50 mM CAPSO.

rUTP-EO-d-TAMRA-2 (4l): 343 μM in 95 μL of 50 mM CAPSO.

rUTP-EO-d-ROX-2 (4m): 174 μM in 55 μL of 50 mM CAPSO.

In the list above, EO represents an propargyl-ethyl-oxide-amino linkerand PA represents a propargyl amine linker. The dyes are abbreviated asfollows: R6G (rhodamnine 6G), R6G-2 (rhodamine 6G-2), R110 (rhodamine110), ROX (rhodamine X), ROX-2 (rhodamine X-2), and TAMRA(tetramethylrhodamine).

Example 9

Energy Transfer Dye-Nucleoside Coupling: General Procedure. See U.S.Pat. No. 5,945,526 to Lee et al.

Approximately 0.6 μmol of ribonucleotide triphosphate in 0.1 M TEAB wasconcentrated to dryness with a centrifuge-vacuum apparatus (e.g., aSpeed-Vac). The resulting solid was dissolved in sodium bicarbonate (250mM, 50 mL) adjusted to pH 9 with 1.0 M NaOH followed by the addition ofthe 5-carboxyfluorescein-NHS ester (FAM-NHS ester) for rGTP-EO-Energytransfer dye or 6-FAM-NHS ester for all other energy transfer dye pairs(1.6 μmol, 10 μL, conc.=5 mg/60 μL DMSO, 2.7 eq), mixed, and allowed tostand for 2-17 h. The reaction mixture was briefly centrifuged to removeparticulates and the reaction mixture was purified via ion exchangefollowed by RP—C8 HPLC as described in Example 1. The resultingFAM-labeled ribonucleotide triphosphate was concentrated and the Fmocmoiety was removed by dissolving the product in concentrated NH₄OH (300μL) and heating at a temperature between 55-60° C. for 0.5 h.

The deprotected FAM-labeled nucleotide was purified via RP—C8 HPLC,concentrated to dryness, and coupled to the appropriatedichlororhodamine (e.g. rUTP-EO-6-FAM-dTAMRA 4o, rCTP-EO-6-FAM-dROX 3p,rATP-PA-6-FAM-dR6G 1j, and rGTP-EO-5-FAM-dR110 2n). Except forA-PA-6-FAM-dR6G, the energy transfer dye coupling was performed insodium bicarbonate buffer (0.25 M, pH=9 (adjusted with NaOH)). For thesynthesis of A-PA-6-FAM-dR6G 1j, bis(trifluoroacetamido)-dR6G-NHS esterwas used. This protected dye is not soluble in aqueous buffer; hence thereactions were performed in freshly-distilled formamide. A 5 μL portionwas diluted with 0.1 M TEAB (500 μL) and a UV spectrum was obtained. Theconcentration (mM) is calculated from Equation 2 above, with thefollowing results:

rATP-PA-6-FAM-d-R6G (1m): 125 μM in 100 mM TEAB.

rGTP-EO-5-FAM-d-R110 (2m): 106 μM in 100 mM TEAB.

rCTP-EO-6-FAM-d-ROX (3m): 71 μM in 100 mM TEAB.

rCTP-EO-6-FAM-d-TAMRA (3o): 138 μM in 100 mM TEAB.

rUTP-EO-6-FAM-d-TAMRA (4m): 144 μM in 100 mM TEAB.

Example 10

This example demonstrates the use of the dye-labeled compounds of theinvention in the direct PCR sequencing of DNA. The twenty microliterreaction mixture comprised 25 mM Tris-Cl, pH 8.8 at 20° C., 1 mMβ-mercaptoethanol, 50 mM KCl, 1.25 mM MgCl₂, 100 μM dATP, 100 μM dCTP,100 μM dGTP, 100 μM dUTP, biotinylated forward primer (200 nM; −21 M13forward primer), reverse primer (200 nM, M13 reverse primer), 50 nMrUTP-PA-6-TAMRA (4i), 50 nM rCTP-PA-6-Rox (3i), 50 nM rGTP-PA-5-R110(2i), 25 nM rATP-PA-6-R6G (1i), 0.75 units of Tma polymerase 25R, 3units of Tma polymerase 30R, 2.5 units of Taq polymerase, and 6 ng oftemplate DNA (pGEM 3Zf(+)).

Tma polymerase 25R and Tma polymerase 30R are described in U.S. Pat. No.5,939,292, which is hereby incorporated by reference herein. The enzymesare, respectively, proofreading and non-proofreading versions of novelthermostable DNA polymerases, which comprise a mutation that increasesthe efficiency of ribonucleotide incorporation. Specifically, eachenzyme has a mutation at Tma codon 678 that changes E678 to glycine.

The reaction mixture was placed in a thermal cycler, heated to 95° C.for 45 seconds, and then subjected to 45 cycles of 95° C. for 15seconds, 55° C. for 30 seconds, and 65° C. for 3 minutes. The finalincubation at 65° C. was continued for an additional 10 minutes. Onemicroliter of the reaction mixture was analyzed on a 2% agarose gel toconfirm that the amplicon was a unique band.

The remaining 19 μl reaction was mixed with 2 μl 250 mM EDTA and 10 μl 1N NaOH and heated to 98° C. for 10 minutes in order to hydrolyze theprimer extension products at sites of dye-labeled rNTP incorporation.The solution was cooled and neutralized by the addition of 10 μl 1 NHCl. Fragments containing the forward primer were captured on 5 μl ofavidin-coupled magnetized beads (Dynabeads®) and washed with 70%ethanol. The captured fragments were eluted from the beads by heating to98° C. for 2 minutes in 3 μl loading buffer (50% formamide (v/v)containing bromophenol blue) and analyzed on an Applied Biosystems 377DNA Sequencer. The sequence determined, which extended through thecomplete multicloning site between the forward and reverse primers basepairs from the forward primer, matched the known sequence of pGEM3Zf(+).

Alternative methods of cleaving the dye-labeledribonucleotide-containing amplicons will be appatent to those skilled inthe art. For example, a mix of various RNAses may be employed. In thiscase, an appropriate amount of a commercially-available blend of RNAses,e.g., RiboShredder® (Epicentre Technologies, Madison, Wis.), is added tothe 20 μl amplification mix and reacted according to the manufacturersprotocols; or a homemade blend of RNAses (RNAses H, A, T1 etc) can bemade, added to the 20 μl of amplification product and reacted at 37° C.for 1-2 hours. Alternatively, amplicons may be cleaved simply byheating, although, the time of heating preferably is increased from the10 minutes used in the alkali/heating protocol to at least 45 minutes at98° C.

Example 11

The dye-labeled ribonucleotides of the invention also may be used todetect SNPs directly in genomic DNA by performing PCR using twooligonucleotide primers that anneal with sequences located sufficientlyclose to each other to provide a small amplicon. To exemplify one suchmethod, which is schematically illustrated in FIG. 1, a twentymicroliter reaction mixture comprising 25 mM Tris-Cl, pH 8.8 at 20° C.,1 mM β-mercaptoethanol, 50 mM KCl, 1.25 mM MgCl₂, 100 μM dATP, 100 μMdCTP, 100 μM dGTP, 100 μM dUTP, 1.25 μM primer A (23 nucleotides inlength), 1.25 μM primer B (21 nucleotides in length), 50 nMrUTP-PA-TAMRA, 50 nM rCTP-PA-Rox, 50 nM rGTP-PA-R110, 25 nMrATP-PA-5R6G, 0.75 units of Tma polymerase 25R, 3 units of Tmapolymerase 30R, 2.5 units of Taq polymerase, and 50 ng of total genomicDNA was made. Primer B was designed so that the first base added to theprimer by the polymerases is the SNP of interest. Because primer A waslonger than primer B, the band corresponding to the SNP position was notcovered by a band from the opposite strand. There was therefore no needto separate the two microsequence ladders prior to electrophoresis.

The reaction mixture was placed in a thermal cycler, heated to 94° C.for 10 minutes, and then subjected to 45 cycles of 94° C. for 15seconds, 55° C. for 5 seconds, and 65° C. for 30 seconds. One microliterof the reaction mixture was analyzed on a 2% agarose gel to confirm thatthe amplicon was a unique band.

The remaining 19 μl reaction was mixed With 2 μl 250 mM EDTA and 10 μl 1N NaOH and heated to 98° C. for 10 minutes in order to hydrolyze theprimer extension products at sites of dye-labeled rNTP incorporation.The solution was cooled and neutralized by the addition of 10 μl 1 NHCl. Loading buffer (20 μl) was added to each sample and 2.5 μl of eachsample were analyzed on an 16% denaturing polyacrylamide gel on anApplied Biosystems 377 DNA Sequencer.

The results of the electrophoretic separation are diagramed in FIG. 1.Because primer B was shorter than primer A, the first band that was seenon the gel corresponded to the SNP on the minus strand of the genomicDNA template, in this case an adenine.

Example 12

This example demonstrates the use of the dye-labeled compounds of theinvention to detect SNPs on both strands of a DNA template in a singlereaction, as illustrated in FIG. 2. The reaction conditions were thesame as those for Example 11, except for the primers, which were 24(primer C) and 26 (primer D) nucleotides in length, and the amount ofgenomic DNA template, 75 ng. Both primers were designed so that thefirst base added to each by the polymerases is the SNP of interest.

The results of the electrophoretic separation of fragments containingthe primers are diagramed in FIG. 2. Because primer C was shorter thanprimer A, the first band that was seen on the gel corresponded to theSNP on the minus strand of the genomic DNA template, in this case anadenine. The first band corresponding to the plus strand was a guanine,indicating that the template was heterozygous for this SNP.

Example 13

The DNA sequence of each strand surrounding a SNP may be unambiguouslydetermined using the compounds of the invention. The reaction conditionsagain are the same as those in Example 11, except that primer E is 18nucleotides in length and primer F comprises 22 nucleotides that arecomplementary to the target DNA and 30 nucleotides that are notcomplementary to the target DNA. Nucleotide 23 of primer F is a modifiednucleotide, for example, an inverted base with a 5′-5′ phosphodiesterbond, that prevents 5′-extension of primer E beyond this point.

The resulting fragments after gel electrophoresis are illustrated inFIG. 3. Extension products of primer E range from 19 to 45 nucleotidesin length, with the SNP present as the 21 nucleotide fragment. Theextension products from primer F, which range in size from 53 to 76nucleotides, are readily separated from the primer E-derived fragments.Again, the fragment identifying the SNP is the third fragment.

The DNA sequence of each strand surrounding a SNP may also beunambiguously determined using the compounds of the invention, if theprimers used are separated after hydrolysis, for example byhybridization to complementary sequences. As diagramed in FIG. 4, primerG comprises hybridization based pull-out (HBP) sequence 1 and primer Hcomprises HBP sequence 2. After hydrolysis with base, the fragmentscomprising primers G and H can be separated from other fragments andfrom each other by hybridization to affinity matrices on whichcomplementary oligonucleotides are immobilized by techniques known tothose skilled in the art, for example, those disclosed in U.S. Pat. No.6,124,092. After the fragments are eluted from the affinity matrices,the sequence of each strand is determined by gel electrophoresis.

Example 14

The dye-labeled compounds of the invention also are useful in animproved method for determining the methylation state of DNA, especiallyof promoter regions in genomic DNA. In this method, a bisulphite salt,preferable sodium bisulfite, is used to convert cytosine residues touracil residues in the target DNA, under conditions whereby5-methylcytosine remains non-reactive. Clark et al., Nucl. Acids Res.22: 2990-97 (1994). The bisulfite-treated DNA is then amplified by PCRwith primers specific for the region of interest in the presence of adye-labeled rCTP analog according to the invention. One microliter ofthe reaction mixture is analyzed on a 2% agarose gel to confirm that theamplicon is a unique band.

The remaining 19 μl reaction is mixed with 2 μl 250 mM EDTA and 10 μl 1N NaOH and heated to 98° C. for 10 minutes in order to hydrolyze theprimer extension products at sites of dye-labeled rCTP incorporation.The solution is cooled and neutralized by the addition of 10 μl 1 N HCl.Loading buffer (20 μl) is added to each sample and 2.5 μl of each sampleis analyzed on an 5% denaturing polyacrylamide gel on an AppliedBiosystems 377 DNA Sequencer. All the cytosine residues remaining in thesequence correspond to methylated cytosines in the template DNA.

Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with the true scope and spiritof the invention being indicated by the following claims.

1. A compound having the formula:

wherein B is a nucleobase; L is a linker selected from apropargyl-ethyl-oxide-amino linker, a propargyl linker, a benzylaminelinker, a rigid linker, a tuned linker, and a heterocycle linker; R₃ istriphosphate, α-thiotriphosphate, or a salt thereof, and Dye is afluorescein-type dye, a rhodamine-type dye, an energy transfer dye pair,or a cyanine-type dye.
 2. The compound according to claim 1, wherein therhodamine-type dye is a 4,7-dichlorophenyl-rhodamine dye.
 3. Thecompound according to claim 2, wherein the 4,7-dichlorophenyl-rhodaminedye is selected from dTAMRA, dROX, dR6G, and dR110.
 4. The compoundaccording to claim 1, wherein the nucleobase is selected from cytosine,adenine, uracil, guanine, 7-deazaadenine, and 7-deazaguanine.
 5. Acompound of the formula II:

wherein L is a linker selected from a propargyl-ethyl-oxide-aminolinker, a propargyl linker, a propargyl-propyl-oxide-amino linker, abenzylamine linker, a rigid linker, and a multimer thereof; wherein R₄is either NH₂, OH, or O, and R₅ is either NH₂, OH, or H; wherein R₃ iseither triphosphate, α-thiotriphosphate, or a salt thereof; and whereinthe dye is any reporter group.
 6. A compound having the formula:

wherein B is a nucleobase selected from uracil, cytosine, adenine,7-deazaadenine, guanine, and 7-deazaguanine; wherein R₃ is triphosphateor a salt thereof; wherein L is a linker selected frompropargyl-ethyl-oxide-amino and propargyl wherein the linker is attachedto the 8-C of a adenine, 7-deazaadenine, guanine, or 7-deazaguaninenucleobase, the 7-C or 8-C of a 7-deazaadenine or 7-deazaguaninenucleobase, or the C-5 of a uracil or cytosine nucleobase; and whereinDye is selected from a rhodamine-type dye and a fluorescein-type dye. 7.The compound according to claim 6, wherein B is uracil; L ispropargyl-ethyl-oxide-amino or propargyl; and Dye is TAMRA or dTAMRA. 8.The compound according to claim 6, wherein B is cytosine; L ispropargyl-ethyl-oxide-amino or propargyl; and Dye is ROX or dROX.
 9. Thecompound according to claim 6, wherein B is 7-deazaadenine; L ispropargyl-ethyl-oxide-amino or propargyl; and Dye is R6G or dR6G. 10.The compound according to claim 6, wherein B is 7-deazaguanine; L ispropargyl-ethyl-oxide-amino or propargylamino; and Dye is R110 or dR110.11. The compound according to claim 6, wherein Dye is an energy transferdye pair comprised of a fluorescein-type dye and a rhodamine-type dyewherein the fluorescein-type dye is attached to the nucleobase through apropargyl-ethyl-oxide-amino or propargyl linker, and thefluorescein-type dye is attached to the rhodamine-type dye by a secondlinker.
 12. The compound according to claim 11, wherein thefluorescein-type dye is 4-aminomethyl FAM and the rhodamine-type dye isa 4,7-dichlororhodamine dye.
 13. The compound according to claim 12,wherein the 4,7-dichlororhodamine dye is dTAMRA, dROX, dR6G, or dR110.